Isolated b7-h4 specific compositions and methods of use thereof

ABSTRACT

The present invention relates to B7-H4-specific compositions and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 14/181,931,filed on Feb. 17, 2014, now allowed, which is a continuation of PCTInternational Application No. PCT/US2012/063546, filed on Nov. 5, 2012,which claims the benefit pursuant to 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/555,406, filed on Nov. 3, 2011, each ofwhich are hereby incorporated by reference in their entirety herein.

BACKGROUND OF THE INVENTION

Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responsesthrough the release of humoral mediators such as cytokines,prostaglandins, growth factors and metalloproteases. TAMs can alsoprotect tumors from immune recognition by hampering cell-mediated immuneresponses through the cell-surface expression of inhibitory moleculessuch as B7-H4 (Kryczek et al., 2006, J Exp Med 203(4):871-81). TAMsderive from resident macrophages or from monocytes recruited by thetumor microenvironment and polarized at the tumor site (Qia and Pollard,2010, Cell 141(1):39-51; Allavena and Mantovani, 2012, Clin Exp Immunol167(2):195-205). Tumor infiltration with TAMs has been associated withpoor patient survival and higher microvessel density (Coffelt et al.,2009, Biochim Biophys Acta 1796(1):11-8). Targeting TAMs represents apromising strategy against cancer and several approaches have alreadybeen developed, including depletion with clodronate liposomes(Zeisberger et al., 2006, Br J Cancer 95(3):272-81); inhibition of tumorrecruitment by targeting of CFSR-1 and CCL2 (Loberg et al., 2007, CancerRes 67(19):9417-24); and “re-education” through activation, via CD40with anti-CD40 mAbs (Beatty et al., 2011, Science 331(6024):1612-6) orvia HRG plasma protein (Rolny et al., 2011, Cancer Cell 19(1):31-44) ormannose receptor (Dangaj et al., 2011, PLoS One 6(12):e28386).

B7-H4 or B7x/B7s is a member of the B7 superfamily and has recentlyidentified as an inhibitory modulator of T-cell response (Sica et al.,2003, Immunity 18(6):849-61; Prasad et al., 2003, Immunity 18(6):863-73;Zang et al., 2003, Proc Natl Acad Sci USA, 100(18):10388-92). Whenpresent at the surface of antigen presenting cells, B7-H4 negativelyregulates T cell activation, possibly through interaction with a ligandthat remains to be identified (Kryczek et al., 2006, J Immunol177(1):40-4). B7-H4 adenoviral overexpression in pancreatic isletsprotected mice from autoimmune diabetes maintaining peripheral tolerance(Wei et al., 2011, J Exp Med, 208(8):1683-94). Consistently with thisobservation, B7-H4 knock-out mice are more resistant to infection byListeria monocytogenes than their wild type littermates due to a higherproliferation of neutrophils in peripheral organs (Zhu et al., 2009,Blood, 113(8):1759-67).

B7-H4 is widely expressed at the mRNA level, but its restricted patternof protein expression in normal tissues suggests posttranscriptionalregulation. B7-H4 expression in tumor tissues was observed in varioustypes of human cancers such as breast (Tringler et al., 2005, ClinCancer Res 11(5):1842-8), ovarian (Kryczek et al., 2006, J Exp Med203(4):871-81), pancreatic, lung (Choi et al., 2003, J Immunol171(9):4650-4; Sun et al., 2006, Lung Cancer 53(2):143-51) melanoma(Quandt et al., 2011, Clin Cancer Res 17(10):3100-11) and renal cellcarcinoma (Jung et al., 2011, Korean J Urol 52(2):90-5; Krambeck et al.,2006, Proc Natl Acad Sci USA 103(27):10391-6). B7-H4 expression wasevaluated by immunohistochemistry in most studies, either as a cytoplasmor a plasma membrane protein (Quandt et al., 2011, Clin Cancer Res17(10):3100-11; Krambeck et al., 2006, Proc Natl Acad Sci USA103(27):10391-6; Jiang et al., 2010, Cancer Immunol Immunother59(11):1707-14; Zang et al., 2007, Proc Natl Acad Sci USA,104(49):19458-63; Miyatake et al., 2007, Gynecol Oncol 106(1):119-27).In ovarian cancer cells, B7-H4 expression was assessed by flow cytometryand was also reported to be mainly intracellular (Kryczek et al., 2006,J Exp Med 203(4):871-81), to the exception of some cell lines where cellsurface expression was observed (Choi et al., 2003, J Immunol171(9):4650-4). B7-H4 has also been detected in a soluble form in bloodsamples from cancer patients (Simon et al., 2006, Cancer Res66(3):1570-5; Thompson et al., 2008, Cancer Res 68(15):6054-8). Thebroad presence in various cancers of a negative regulator of T cellactivation suggests a role of B7-H4 in down-regulating antitumorimmunity. In fact, ovarian cancer-derived B7-H4⁺ TAMS suppress HER2antigen-specific T-cell proliferation and cytotoxicity, and the blockingof B7-H4 expression on macrophages using morpholino antisenseoligonucleotides in vitro and in vivo improves tumor-associated antigenT-cell responses (Kryczek et al., 2006, J Exp Med 203(4):871-81).

Altogether, these results support B7-H4 translational value as a targetmolecule for anti-tumor immunotherapy. However, the use of antisensenucleic acids remains limiting in clinics, in part because of a lowstability in vivo due to serum inactivation, enzyme degradation, andinnate immune activation, but also because of the lack of specifictargeting and rapid elimination when oligonucleotides are delivered in anaked form (Zhang et al., 2012, Mol Ther 20(7):1298-304). Other meansfor blocking B7-H4 activity need to be developed for clinicalapplications. The present invention addresses this need.

SUMMARY OF THE INVENTION

The invention provides an isolated polynucleotide encoding a humananti-B7-H4 antibody or a fragment thereof, wherein the antibody or afragment thereof comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4. In one embodiment, the isolatedpolynucleotide encoding a human anti-B7-H4 antibody or a fragmentthereof comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 5-8.

In one embodiment, the isolated polypeptide encoding a human anti-B7-H4antibody or a fragment thereof comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-4.

In one embodiment, the antibody or fragment thereof comprises a fragmentselected from the group consisting of an Fab fragment, an F(ab′)₂fragment, an Fv fragment, and a single chain Fv (scFv).

The present invention provides a method for diagnosing a disease,disorder or condition associated with the expression of B7-H4 on a cell,the method comprising a) contacting the cell with a human anti-B7-H4antibody or fragment thereof, wherein the antibody or a fragment thereofcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-4; and b) detecting the presence of B7-H4 wherein thepresence of B7-H4 diagnoses for the disease, disorder or conditionassociated with the expression of B7-H4.

In one embodiment, the disease, disorder or condition associated withthe expression of B7-H4 is cancer.

The invention also provides a method of diagnosing, prognosing, ordetermining risk of a B7-H4-related disease in a mammal, the methodcomprising detecting the expression of B7-H4 in a sample derived fromthe mammal comprising: a) contacting the sample with a human anti-B7-H4antibody or fragment thereof, wherein the antibody or a fragment thereofcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-4; and b) detecting the presence of B7-H4 wherein thepresence of B7-H4 diagnoses for a B7-H4-related disease in the mammal.

The invention also provides a method of inhibiting B7-H4-dependent Tcell inhibition, the method comprising contacting a cell with a humananti-B7-H4 antibody or fragment thereof, wherein the antibody or afragment thereof comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4. In one embodiment, the cell isselected from the group consisting of a B7-H4-expressing tumor cell, atumor-associated macrophage (TAM), and any combination thereof.

The invention also provides a method of blocking T-cell inhibitionmediated by a B7-H4-expressing cell and altering the tumormicroenvironment to inhibit tumor growth in a mammal, the methodcomprising administering to the mammal an effective amount of acomposition comprising an isolated anti-B7-H4 antibody or fragmentthereof, wherein the antibody or a fragment thereof comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-4. Inone embodiment, the cell is selected from the group consisting of aB7-H4-expressing tumor cell, a tumor-associated macrophage (TAM), andany combination thereof.

The invention also provides a method of inhibiting, suppressing orpreventing immunosuppression of an anti-tumor or anti-cancer immuneresponse in a mammal, the method comprising administering to the mammalan effective amount of a composition comprising an isolated anti-B7-H4antibody or fragment thereof, wherein the antibody or a fragment thereofcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-4. In one embodiment, the antibody or fragment thereofinhibits the interaction between a first cell with a T cell, wherein thefirst cell is selected from the group consisting of a B7-H4-expressingtumor cell, a tumor-associated macrophage (TAM), and any combinationthereof.

The invention also provides an isolated nucleic acid sequence encoding achimeric antigen receptor (CAR), wherein the isolated nucleic acidsequence comprises the sequence of a human B7-H4 binding domain and thesequence of a CD3 zeta signaling domain. In one embodiment, isolatednucleic acid sequence further comprises the sequence of a co-stimulatorysignaling domain.

In one embodiment, the co-stimulatory signaling domain is selected fromthe group consisting of the CD28 signaling domain, the 4-1BB signalingdomain, and any combination thereof.

In one embodiment, the human B7-H4 binding domain is a human antibody ora fragment thereof is selected from the group consisting of an Fabfragment, an F(ab′)₂ fragment, an Fv fragment, and a single chain Fv(scFv).

In one embodiment, the antibody or a fragment thereof comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-4. Inanother embodiment, the antibody or a fragment thereof comprises anucleic acid sequences selected from the group consisting of SEQ ID NOs:5-8.

The invention also provides an isolated chimeric antigen receptor (CAR)comprising a human B7-H4 binding domain and a CD3 zeta signaling domain.In one embodiment, the CAR further comprises the sequence of aco-stimulatory signaling domain.

In one embodiment, the co-stimulatory signaling domain is selected fromthe group consisting of the CD28 signaling domain, the 4-1BB signalingdomain, and any combination thereof.

In another embodiment, the human B7-H4 binding domain is a humanantibody or a fragment thereof is selected from the group consisting ofan Fab fragment, an F(ab′)₂ fragment, an Fv fragment, and a single chainFv (scFv). In one embodiment, the antibody or a fragment thereofcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-4.

The invention also provides a method of providing an anti-tumor immunityin a mammal, the method comprising administering to the mammal aneffective amount of a genetically modified cell comprising an isolatednucleic acid sequence encoding a chimeric antigen receptor (CAR),wherein the isolated nucleic acid sequence comprises the sequence of ahuman B7-H4 binding domain and the nucleic acid sequence of a CD3 zetasignaling domain.

In one embodiment, the cell is an autologous T cell.

In one embodiment, the mammal is a human.

The invention also provides a method of treating a mammal having adisease, disorder or condition associated with dysregulated expressionof B7-H4, the method comprising administering to the mammal an effectiveamount of a genetically modified cell comprising an isolated nucleicacid sequence encoding a chimeric antigen receptor (CAR), wherein theisolated nucleic acid sequence comprises the sequence of a human B7-H4binding domain and the nucleic acid sequence of a CD3 zeta signalingdomain.

In one embodiment, the disease, disorder or condition associated withdysregulated expression of B7-H4 is selected from the group consistingof liver cancer, pancreatic cancer, ovarian cancer, stomach cancer, lungcancer, endometrial cancer, hepatocellular carcinoma, and anycombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1B are a series of images showing flow cytometry analysis ofB7-H4 cell surface expression on ovarian cancer samples. PE-labeledanti-B7-H4 mAb staining of CD45-EpCam+ tumor cells (FIG. 1A) andCD45+CD14+ monocytes (FIG. 1B) from 5 ovarian cancer patients.

FIGS. 2A-2D are a series of images showing upregulation of B7-H4 onmacrophages in vitro. FIG. 2A is an image of the morphology of M1 and M2macrophages after in vitro cytokine maturation. FIGS. 2B-2D demonstratethat induction of macrophage B7-H4 expression was determined by (FIG.2B) qRT-PCR; and (FIGS. 2C-2D) flow cytometry, after (FIG. 2B lanes 1-2and FIG. 2C) cytokine stimulation, or (FIG. 2B lane 3 and FIG. 2D)co-culture with Ovcar3 tumor cell line.

FIGS. 3A-3B are a series of images showing validation of anti-B7-H4scFv. FIG. 3A demonstrates anti-B7-H4 scFv binding to serial dilutionsof recombinant B7-H4 vs. irrelevant antigen as measured by captureELISA. FIG. 3B is an image of a flow cytometry analysis of anti-B7-H4biobodies binding to M1 and M2 macrophages after pre-coupling tofluorescent streptavidin beads (Myltenyi).

FIGS. 4A-4E are a series of images demonstrating blocking ofB7-H4-mediated T cell suppression using anti-B7-H4 scFvs in vitro. FIGS.4A-4D demonstrate that T cell proliferation was assessed after analyzingCFSE dilution in anti-CD3/CD28 stimulated T cells alone (FIG. 4A); Tcells with M1 macrophages (FIG. 4B); T cells in transwell co-culturewith ovcar3 (FIG. 4C); T cells with TAMs in transwell co-culture withovcar3 (FIG. 4D). FIG. 4E demonstrates that T cell activation wasevaluated based on CD69 expression after anti-CD3/CD28 stimulation of Tcells alone or in presence of M1 or TAMs.

FIGS. 5A-5D are a series of images that demonstrate B7-H4 expression inhuman ovarian cancer cells. FIG. 5A is an image of a flow cytometry andwestern blot analysis of B7-H4 expression in ovarian cancer cell lines.Cell lines (as indicated) were labeled with PE-conjugated anti-B7-H4 mAb(open histogram) or PE-conjugated isotype control (grey filledhistogram). EBV B cells, M2 macrophages and B7-H4 transduced C3O cellswere used as positive control (upper panel). The lower panel shows aWestern blot analysis of B7-H4 expression in four ovarian cancer celllines (OVCAR5, SKOV3, A1857, OVCAR3). IL4-IL10 in vitro maturatedmacrophages (M2 MΦ) were used as positive controls for B7-H4 expression.β-actin detection was used as endogenous protein loading control. FIG.5B shows a flow cytometry evaluation of B7-H4 surface expression inhuman ovarian cancer ascites and solid tumors samples. Dead cells wereexcluded from total cells based on 7-AAD uptake. Tumor cells andleukocytes were distinguished through the detection of Epcam and CD45surface markers. Macrophage population that expressed surface B7-H4 werecharacterized by the co-expression of CD45⁺CD14⁺CD206⁺B7-H4⁺. Tumorcells that expressed surface B7-H4 were characterized by theco-expression of Epcam⁺B7-H4⁺. FIGS. 5C-5D depict B7-H4 cell surfaceexpression on tumor cells in ascites and solid tumor samples derivedfrom a xenogeneic mouse model of human ovarian cancer. FIG. 5C shows arepresentative flow cytometry analysis of B7-H4 cell surface expressionon freshly harvested, uncultured Epcam⁺ tumor cells (upper panels) vs.Epcam⁺ tumor cells after short in vitro culture (lower panels). FIG. 5Dis an image of a plot of percentages of B7-H4+EpCAM+ cells derived fromascites and solid tumors before and after short term culture, asmeasured by flow cytometry (n=6). Isotype PE IgG1 was used as stainingcontrol for B7-H4.

FIGS. 6A-6C are a series of images demonstrating the isolation ofanti-B7-H4 scFvs from a new yeast display library of ovarian cancerpatients and validation. FIG. 6A is a schematic representation ofprotein-based (FIG. 6B) and cell-based isolation (FIG. 6C) strategies.Protein-based and cell-based isolated anti-B7-H4 scFvs wereplastic-immobilized and incubated with serial dilutions of biotinylatedrecombinant B7-H4 (black diamonds) or irrelevant control antigen (BSA,grey triangles) (FIGS. 6B-6C). Protein binding to scFv was detected withSA-HRP. Colorimetric signal was developed with TMB substrate solution,quenched with sulfuric acid and read at 450 nm on a Biotek ELISA reader.

FIGS. 7A-7D are a series of images demonstrating that recombinant B7-H4protein inhibits polyclonal T cell stimulation and anti-B7-H4 scFvsreverse T cell inhibition. T cells were stimulated with plate boundanti-CD3 and/or anti-CD28 in the presence of immobilized recombinantB7-H4 (black bars) or irrelevant control protein (FOLR1, grey bars).FIG. 7A depicts IFN-γ production (ELISA assays). p=0.032 forCD3-stimulated T cells and p=0.097 for CD3/CD28-stimulated T cells inthe absence or in the presence of B7-H4. Error bars represent standarderror of mean (SEM). FIG. 7B depicts a flow cytometry analysis of CD69 Tcell expression and T cell proliferation as measured by CFSE dilutions,as indicated. FIG. 7C illustrates IFN-γ production (ELISA assays) from Tcells stimulated with plate bound anti-CD3 in the presence ofimmobilized recombinant B7-H4 (black bars) or of irrelevant controlprotein (FOLR1, grey bars) in regular medium (untreated) or in thepresence of anti-B7-H4 scFvs clones #26, #56, 3#54, or 3#68, asindicated. Untreated samples, p=0.0014; #26, p=0.0019; #56, p<0.0001;3#54 p=0.0406; 3#68, p=0.1305. Error bars represent standard error ofmean (SEM). FIG. 7D depicts a flow cytometry analysis of CD69 T cellexpression and T cell proliferation as measured by CFSE dilutions, asindicated.

FIGS. 8A-8E are a series of images demonstrating that B7-H4⁺ APCsinhibit antigen-specific T cells and anti-B7-H4 scFvs reverse T cellinhibition. FIG. 8A depicts a flow cytometry analysis of B7-H4 surfaceexpression in wild type or B7-H4 transduced T2 APCs using PE anti-B7-H4mAb (open histogram) or isotype PE (grey filled histogram). IFN-γproduction of HER-2 (FIG. 8B) and MART-1 (FIG. 8C) TCR specific T cellsafter stimulation with B7-H4 transduced T2 APCs (T2 B7-H4, black bars)or wild type T2 (T2, grey bars) pulsed with MART-1 or HER-2 peptides.p=0.0362 for HER-2 TCR T cells stimulated with HER-2-pulsed T2 vs T2B7-H4; p=0.0024 for MART-1 TCR T cells stimulated with MART-1-pulsed T2vs T2 B7-H4; IFN-γ production of HER-2 (FIG. 8D) and MART-1 (FIG. 8E)TCR specific T cells after stimulation with B7-H4 expressing T2 APCs (T2B7-H4, black bars) or GFP transduced T2 APCs (GFP T2, dark grey bars),pulsed with HER-2 (FIG. 8D) or MART-1 (FIG. 8E) peptides, in thepresence of anti-B7-H4 scFvs, as indicated, or in regular medium(untr.). One Way Anova analysis for IFNγ production of antigen-specificT cells stimulated by peptide-loaded GFP T2 APCs, p=0.7893 (FIG. 8D,dark grey bars) and p=0.2931 (FIG. 8E, dark grey bars). One Way Anovaanalysis for IFNγ production of antigen-specific T cells stimulated bypeptide-loaded T2 B7-H4 APCs, p=0.0066 (FIG. 8D, black bars) andp<0.0001 (FIG. 8E, black bars). In presence of anti-B7-H4 scFvs 3#68,p=0.5748 for HER-2 TCR T cells stimulated by peptide-loaded GFP vs. T2B7-H4 APCs (FIG. 8D) and p=0.2892 for MART-1 TCR T cells stimulated bypeptide-loaded GFP vs. T2 B7-H4 APCs (FIG. 8E). Error bars representstandard error of mean (SEM).

FIGS. 9A-9C are a series of images demonstrating that B7-H4⁺ HER2⁺ tumorcells inhibit HER2-TCR transduced T cell activation and anti-B7-H4 scFv3#68 overcomes T cell inhibition. FIG. 9A depicts a flow cytometryanalysis of B7-H4 surface expression in wild type 624 (WT 624) or B7-H4transduced HLA A2⁺HER2⁺ 624 (B7-H4⁺ 624) cells, and wild type (WTMDA231) or B7-H4 transduced HLA A2^(high) HER2⁺ MDA231 (B7-H4⁺ MDA231)cells, with PE anti-B7-H4 mAb (open histogram) or isotype PE (greyfilled histogram). FIG. 9B depicts IFN-γ secretion of HER-2 TCR specificT cells after stimulation with WT 624 vs. B7-H4+624 (N/A), and with WTMDA231 vs. B7-H4 MDA231 (p=0.0451). FIG. 9C depicts IFN-γ secretion ofHER-2 TCR specific T cells after stimulation with WT MDA231 vs. B7-H4MDA231 in the presence of anti-B7-H4 scFvs, as indicated. One Way Anovaanalysis for IFNγ production of antigen-specific T cells stimulated byWT MDA231, p=0.1726 (dark grey bars). One Way Anova analysis for IFNγproduction of antigen-specific T cells stimulated by B7-H4+ MDA231,p=0.0066 (black bars). With anti-B7-H4 scFv 3#54 stimulation with WT vs.B7-H4+ MDA231, p=0.4393; with anti-B7-H4 scFv 3#68 stimulation with WTvs. B7-H4+ MDA231, p=0.2179. Error bars represent standard error of mean(SEM).

FIGS. 10A-10B are a series of images demonstrating that B7-H4⁺ TAMsinhibit antigen-specific T cells and anti-B7-H4 scFvs reverse T cellinhibition. FIG. 10A depicts IFN-γ production of MART-1 TCR specific Tcells in the presence (black bars) or in the absence (grey bars) ofB7-H4⁺ TAMs during stimulation with T2 APCs pulsed with serial dilutionsof MART-1 peptide or constant concentration (1 μM) of HER-2 peptide tocontrol for specific MART-1 TCR T cell stimulation. At dilution 0.0025μM, p=0.0287; at dilution 0.05 μM, p=0.2777; at dilution 1 μM, p=0.1268.FIG. 10B depicts IFN-γ production of MART-1 TCR specific T cells in thepresence (black bars) or in the absence (grey bars) of B7-H4⁺ TAMsduring stimulation with T2 APCs pulsed with MART-1 peptide in thepresence of anti-B7-H4 scFvs, as indicated. ANOVA to calculate. Errorbars represent standard error of mean (SEM).

FIGS. 11A-11C depict cloning, expression, and purification ofrecombinant B7-H4. FIG. 11A depicts cDNA expression of B7-H4 inmacrophages (1) after 2 and 5 hrs of stimulation with IL10/IL4 or (2)after 72 hrs in transwell co-culture with OVCAR3 cell line. Simultaneousβ-actin amplification was used as control. FIG. 11B is a schematic ofmammalian cloning vector (pTT28) encoding soluble recombinant B7-H4protein fused to a 6×HIS Tag. FIG. 11C depicts detection of recombinantB7-H4 (1) by western blot in the supernatant of 293-F cells using aHIS-probe followed by anti-mouse HRP or (2) after purification using ananti-human B7-H4 goat polyclonal Ab followed by polyclonal rabbitanti-goat HRP; (3) by coumassie staining after electrophoresis.

FIGS. 12A-12B are a series of images demonstrating that B7-H4⁺ TAMsdownregulate antigen-specific T cells proliferation and co-stimulation.MART-1 TCR specific T cells were stimulated with wild type T2 APCspulsed with serial dilutions (0.0025-1 uM) of MART-1 or constantconcentration (1 uM) of HER-2 irrelevant peptide to control for specificMART-1 TCR T cell stimulation. Proliferation was analyzed by CFSEdilution (FIG. 12A); co-stimulation was analyzed by detection of CD137expression (FIG. 12B).

FIG. 13 is an image demonstrating that T cells bearing B7-H4 CARscomprised of scFvs bind human and recombinant B7-H4 with differentaffinity.

FIG. 14 is an image demonstrating that CARs bind both mouse and humanB7-H4 proteins.

FIGS. 15A-15B are series of images demonstrating that T cells bearingB7-H4 CARs specifically react against B7H4+ cells. FIG. 15A demonstratesthat B7-H4 CARs specifically react against B7H4+ ovarian cancer cells.FIG. 15B demonstrates differential B7H4 scFv CAR recognition of varyingB7H4-expressing solid tumor cell lines and a lymphoma tumor cell line.

FIGS. 16A-16B are a series of images demonstrating the reactivity ofB7-H4 CAR transduced T cells. FIG. 16A is an image demonstrating thatB7-H4 CAR transduced T cells are reactive against macrophages expressingdifferential levels of B7H4. FIG. 16B is an image demonstrating that #68B7H4 CAR is comparable to the well-established anti-CD19 CAR in responseto the respective tumor antigen.

FIGS. 17A-7B are a series of images demonstrating the inhibition ofB7-H4 CARs. FIG. 17A is a set of images demonstrating that B7-H4 CAR Tcells are not inhibited by immobilized B7-H4 protein. FIG. 17B is animage depicting that the addition of soluble #68scFv to the culturespecifically inhibits #68 B7H4 CAR activity (IFNγ secretion) againstimmobilized B7-H4 protein, thus demonstrating the B7-H4 CAR isspecifically blocked.

FIG. 18 is an image demonstrating that the addition of 68 scFv in thepresence of CD3 leads to specific inhibition of 68 B7-H4 CAR IFN-γsecretion and specific moderate rescue of 56 B-7H4 CAR IFN-γ secretion.

FIG. 19 is an image demonstrating that 68scFv B7H4 CAR MIP1a cytokinesecretion is not diminished as a result of B7H4 CAR-B7H4 Ag signaling.

DETAILED DESCRIPTION

The present invention is based partly on the identification ofhuman-derived antibodies that target B7-H4. The antibodies of theinvention are used for diagnostic and in vivo therapeutic applications.In one embodiment, the antibodies of the invention specifically bind toB7-H4.

In another embodiment, the antibodies of the invention block theinhibition of T cell proliferation. In yet another embodiment, theanti-B7-H4 antibodies of the invention reverse T-cell inhibitionmediated by a B7-H4 signalling. In one embodiment, the antibodies blockB7-H4-dependent T cell inhibition.

In another embodiment, the antibodies of the invention restore T cellproliferation against tumor cells in the presence of macrophages andhence are a useful therapeutic composition against cancer. For example,blocking B7-H4 using an antibody of the invention overcomesantigen-specific T cell inhibition mediated by B7-H4 expressed on thetumor cell surface.

In one embodiment, the antibodies of the invention are scFv antibodies.In some embodiments, the antibodies of the invention are used fordiagnosing the presence of B7-H4 in a biological sample.

In one embodiment, the antibodies of the invention are used for therapyagainst a disease, disorder or condition associated with dysregulationof B7-H4 expression. In one embodiment, the antibodies of the inventionare used for cancer therapy against cancers associated with dysregulatedexpression of B7-H4.

The present invention relates generally to the treatment of a patienthaving a cancer associated with dysregulated expression of B7-H4, or atrisk of having a cancer associated with dysregulated expression ofB7-H4, using cellular infusion. In one embodiment, lymphocyte in fusion,preferably autologous lymphocyte infusion is used in the treatment.

In one embodiment, PBMCs are collected from a patient in need oftreatment and T cells therefrom are engineered and expanded using themethods described herein and then infused back into the patient. Theinvention is not limited to a particular cell or cell type. Rather, anycell or cell type can be engineered and expanded using the methodsdescribed herein and then infused back into the patient.

The present invention also relates generally to the use of T cellsengineered to express a Chimeric Antigen Receptor (CAR). CARs combine anantigen recognition domain of a specific antibody with an intracellularsignaling molecule. For example, in some embodiments, the intracellularsignaling molecule includes, but is not limited to, CD3-zeta chain,4-1BB and CD28 signaling modules and combinations thereof. Preferably,the antigen recognition domain binds to B7-H4. More preferably, theantigen recognition domain comprises a fully human anti-B7-H4.Accordingly, the invention provides a fully human anti-B7-H4-CARengineered into a T cell and methods of their use for adoptive therapy.

In one embodiment, the invention includes autologous cells that aretransfected with a vector comprising a fully-human anti-B7-H4 CARtransgene. Preferably, the vector is a retroviral vector. Morepreferably, the vector is a self-inactivating lentiviral vector asdescribed elsewhere herein.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv) andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NewYork; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

As used herein, the term “B7-H4” refers to B7-H4 from any mammalianspecies and the term “hB7-H4” refers to human B7-H4. Further details onB7-H4 polypeptides and nucleic acids are provided in U.S. Pat. No.6,891,030, the disclosure of which is incorporated herein by referencein its entirety. The nucleotide and amino acid sequences of hB7-H4 canbe found in GenBank under Accession Nos. AY280972 and AAP37283,respectively. B7-H4 is a negative regulator of T cell-mediated immunity.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for the abilityto bind B7-H4 using the functional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

The term “dysregulated” when used in the context of the level ofexpression or activity of B7-H4 refers to the level of expression oractivity that is different from the expression level or activity ofB7-H4 in an otherwise identical healthy animal, organism, tissue, cellor component thereof. The term “dysregulated” also refers to the alteredregulation of the level of expression and activity of B7-H4 compared tothe regulation in an otherwise identical healthy animal, organism,tissue, cell or component thereof

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. Such results may include, butare not limited to, the inhibition of virus infection as determined byany means suitable in the art.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the plasma membrane of a cell. An example ofa “cell surface receptor” is human B7-H4.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals).

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,lentiviral vectors, and the like.

By the term “specifically binds,” as used herein, is meant an antibody,or a ligand, which recognizes and binds with a cognate binding partner(e.g., a stimulatory and/or costimulatory molecule present on a T cell)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides isolated antibodies, particularly humanantibodies that bind specifically to B7-H4. In certain embodiments, theantibodies of the invention comprise particular structural features suchas CDR regions comprising particular amino acid sequences. The inventionalso provides methods of making such antibodies.

In one embodiment, the invention provides a number of antibodies orfragments thereof engineered for enhanced binding to a B7-H4 proteinexpressed on a cell surface. In another embodiment such antibodyfragments are functional in that they provide a biological responseincluding but is not limited to, activation of an immune response,inhibition of signal-transduction origination from its target antigen,inhibition of kinase activity, and the like, as will be understood by askilled artisan. Preferably, the antibodies and fragments thereof of theinvention can block the inhibition of T cell proliferation. In yetanother embodiment, the anti-B7-H4 antibodies of the invention canreverse T-cell inhibition mediated by a B7-H4 signalling. In yet anotherone embodiment, the antibodies can block B7-H4-dependent T cellinhibition.

In some embodiments, the antibodies of the invention are incorporatedinto an immunoconjugate, a chimeric antigen receptor (CAR), apharmaceutical composition, and the like. Accordingly, the presentinvention provides compositions and methods for treating, among otherdiseases, cancer or any malignancy or autoimmune disease in whichexpression of B7-H4 is dysregulated.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a chimeric antigen receptor (CAR) wherein the CART cell exhibits an antitumor property. A preferred antigen is B7-H4. Inone embodiment, the antigen recognition domain of the CAR comprises afully human anti-B7-H4. Accordingly, the invention provides a fullyhuman anti-B7-H4-CAR engineered into a T cell and methods of their usefor adoptive therapy.

In one embodiment, the anti-B7-H4-CAR comprises one or moreintracellular domain selected from the group of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD3zeta signal domain, andany combination thereof. This is because the present invention is partlybased on the discovery that CAR-mediated T-cell responses can be furtherenhanced with the addition of costimulatory domains.

Anti-B7-H4 Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to human B7-H4. In one embodiment, theinvention relates to an isolated human antibody or functional fragmentthereof, wherein the antibody specifically binds to a B7-H4 protein orfragment thereof, wherein the antibody or functional fragment thereof isencoded by an amino acid sequence comprising a sequence selected fromthe group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 2.

In one embodiment, the anti-B7H4 scFv clone 56 polypeptide is encoded bythe amino acid sequence set forth in SEQ ID NO: 1. In anotherembodiment, the nucleic acid sequence encoding the anti-B7H4 scFv clone56 polypeptide is set forth in SEQ ID NO: 5.

In one embodiment, the anti-B7H4 scFv clone 26 polypeptide is encoded bythe amino acid sequence set forth in SEQ ID NO: 2. In anotherembodiment, the nucleic acid sequence encoding the anti-B7H4 scFv clone26 polypeptide is set forth in SEQ ID NO: 6.

In one embodiment, the anti-B7H4 scFv clone 3#54 (also referred hereinas anti-B7H4 scFv clone 54) polypeptide is encoded by the amino acidsequence set forth in SEQ ID NO: 3. In another embodiment, the nucleicacid sequence encoding the anti-B7H4 scFv clone 3#54 polypeptide is setforth in SEQ ID NO: 7.

In one embodiment, the anti-B7H4 scFv clone 3#68 (also referred hereinas anti-B7H4 scFv clone 54) polypeptide is encoded by the amino acidsequence set forth in SEQ ID NO: 4. In another embodiment, the nucleicacid sequence encoding the anti-B7H4 scFv clone 3#68 polypeptide is setforth in SEQ ID NO: 8.

In another embodiment, the invention relates to a recombinant B7-H4protein, wherein the recombinant protein is encoded by the amino acidsequence comprising the sequence set forth in SEQ ID NO: 9. In anotherembodiment, the nucleic acid sequence encoding the recombinant B7-H4protein is set forth in SEQ ID NO: 10.

In one embodiment, the antibody fragment provided herein is a singlechain variable fragment (scFv). In another embodiment, the antibodies ofthe invention may exist in a variety of other forms including, forexample, Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e.bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)). In one embodiment, the antibodies andfragments thereof of the invention binds a B7-H4 protein with wild-typeor enhanced affinity.

In one embodiment, the anti-B7-H4 scFvs provided herein are from humanorigin. In another embodiment, the anti-B7-H4 scFvs provided herein wereisolated from a yeast-display library of antibody fragments generatedfrom B cells isolated from ascites of ovarian cancer patients. Inanother embodiment, the anti-B7-H4 scFvs are screened using arecombinant B7-H4 protein such as one exemplified by SEQ ID NO: 9. Inanother embodiment, the anti-B7-H4 scFvs provided herein are tested inin vitro co-culture model systems of macrophages, T cells and tumorcells. In another embodiment, the anti-B7-H4 scFvs provided herein havethe advantage that they can bind B7-H4 expressed on the surface of both,monocytes and tumor cells such as macrophages and ovarian cancer cells.

In one embodiment, an antibody of the invention comprises heavy andlight chain variable regions comprising amino acid sequences that arehomologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-B7-H4 antibodies of the invention.

In some embodiments, the antibody of the invention is further preparedusing an antibody having one or more of the VH and/or VL sequencesdisclosed herein can be used as starting material to engineer a modifiedantibody, which modified antibody may have altered properties ascompared to the starting antibody. In various embodiments, the antibodyis engineered by modifying one or more amino acids within one or bothvariable regions (i.e., VH and/or VL), for example within one or moreCDR regions and/or within one or more framework regions. Additionally oralternatively, an antibody is engineered by modifying residues withinthe constant region(s), for example to alter the effector function(s) ofthe antibody.

Methods

In one embodiment, scFvs provided herein are used to detect or targetB7-H4, thus permitting diagnostic tests in vitro and/or in vivo(imaging), as well as the production of targeted therapeutics. In someinstances, the antibodies of the invention are coupled with therapeuticreagents, for example, coupled onto nanoparticles with payload, or as achimeric antigen receptor (CAR) for T cell therapy. Hence, because ofits functional properties in vitro, scFvs provided herein can also beused in vivo as a naked reagent to boost anti-tumor immunity.

In one embodiment, the invention relates to a method of diagnosing aB7-H4-related disease in a subject, the method comprising the step of a)administering to a subject an effective amount of composition comprisingan anti-B7-H4 antibody or fragment thereof operably linked to a labelingagent, b) obtaining a biological sample from the subject, c) detectingbinding of the composition to the biological sample, d) whereindetecting the binding of the composition to the biological sample fromthe subject is indicative of the subject having a B7-H4-related disease,and wherein the antibody or fragment thereof is encoded by the aminoacid sequence comprising SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4.

In another embodiment, the invention relates to a method of diagnosing aB7-H4-related disease in a subject, the method comprising the step of a)obtaining a biological sample from the subject b) contacting the samplewith an effective amount of a composition comprising an anti-B7-H4antibody or fragment thereof operably linked to a labeling agent, c)detecting binding of the composition to the biological sample, d)wherein detecting the binding of the composition to the biologicalsample from the subject is indicative of the subject having aB7-H4-related disease, and wherein the antibody or fragment thereof isencoded by the amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO: 4.

In one embodiment, the invention includes a method of inhibiting,suppressing or preventing immunosuppression of an anti-tumor oranti-cancer immune response in a subject, the method comprisingadministering to the subject an effective amount of a compositioncomprising an isolated anti-B7-H4 antibody of fragment thereof, whereinthe antibody or fragment thereof is encoded by the amino acid sequencecomprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Inanother embodiment, the inhibition of immunosuppression prevents theinteraction between macrophages expressing B7-H4 protein and T cellsthat would otherwise function to effect an anti-tumor response.Therefore, in another embodiment, inhibiting the interaction betweenmacrophages and T cells inhibits immunosuppresion of the subject'santi-tumor immune response.

In one embodiment, the invention includes a method of treating a canceror a tumor growth, the method comprising the step of administering to asubject an effective amount of a composition comprising an isolatedanti-B7-H4 antibody or fragment thereof, wherein administering thecomposition blocks a tumor-associated macrophage- or tumor cell-mediatedimmunosuppression of an anti-tumor response and enables activation of ananti-tumor response, wherein the antibody or fragment thereof is encodedby the amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 4.

In one embodiment, the invention provides a method of preventing acancer metastasis, the method comprising the step of administering to asubject a composition comprising an anti-B7-H4 antibody or fragmentthereof, wherein administering the antibody or fragment thereof blocks atumor-associated macrophage- or tumor cell-mediated immunosuppression ofan anti-tumor response and prevents the cancer metastasis, wherein theantibody or fragment thereof is encoded by the amino acid sequencecomprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In another embodiment, the invention provides a method of blockingT-cell inhibition mediated by a B7-H4-expressing cell and altering thetumor microenvironment to inhibit tumor growth in a subject, the methodcomprising the step of administering to the subject an effective amountof a composition comprising an isolated anti-B7-H4 antibody or fragmentthereof, wherein the antibody or fragment thereof is encoded by theamino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,or SEQ ID NO: 4.

In one embodiment, provided herein is a method of down-regulating theexpression of a B7-H4 receptor or protein on the cell surface comprisingcontacting the receptor or protein with an B7-H4-specific antibody orfragment thereof, wherein the antibody or fragment thereof is encoded bythe amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 4. It is to be understood by a skilled artisan thata specifically desired level of down-regulation can be achieved bycontacting the B7-H4 receptor or protein provided herein, with anempirically determined dose of the anti-B7-H4 antibody or fragmentthereof. In another embodiment, the term “downregulation” refers toinhibition or reduction of expression of the cell-surface B7-H4 protein,resulting in a preventive, diagnostic or therapeutic effect. In anotherembodiment, the down-regulation is two-fold, three-fold, five-fold orhigher. In another embodiment, the signaling pathway starting at theoncogenic receptor that leads to the expression of more oncogenicreceptor is down-regulated upon contacting the receptor with theantibody or fragment thereof of the invention.

In one embodiment, the invention provides a method of blocking aninteraction between a receptor and a ligand comprising contacting thereceptor with an B7-H4-specific antibody or fragment thereof, whereinthe antibody or fragment thereof is encoded by the amino acid sequencecomprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Inanother embodiment, inhibiting the interaction amongst tumor cells andmacrophages that express B7-H4 on their surface, and T cells, inhibitsimmunosuppresion of the subject's anti-tumor immune response. In anotherembodiment, the immune response is a cell-mediated anti-tumor immuneresponse, where in another embodiment the immune response is a T-cellmediated immune response. In another embodiment, the T-cell mediatedimmune response is a CD4 or a CD8 T-cell immune response.

In one embodiment, the invention provides a method of delivering abiologically active agent to a cell expressing B7-H4 on its surface, themethod comprising contacting the cell with a bioconjugate compositioncomprising an anti-B7-H4 antibody or fragment thereof operably linked tothe biologically active agent, wherein the antibody or fragment thereofhas an amino acid sequence encoded by the amino acid sequence comprisingSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In anotherembodiment, the method further comprises the delivery of one or morebiologically active agents. In another embodiment, the biologicallyactive agent is a cytotoxic agent, a chemotherapeutic agent, a cytokine,a growth inhibitory agent, an anti-hormonal agent, a kinase inhibitor,an anti-angiogenic agent, a cardioprotectants, a toxin, a radioisotope,or a combination thereof.

In one embodiment, the method provides a method of labeling a cellexpressing B7-H4 on its cell surface, the method comprising the step ofcontacting the cell with a bioconjugate composition comprising ananti-B7-H4 antibody or fragment thereof operably linked to a labelingagent, wherein the antibody or fragment thereof is encoded by an aminoacid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, orSEQ ID NO: 4. In another embodiment, the labeling is carried out invivo. In another embodiment, the labeling enables imaging the cell,where in another embodiment, the labeling enables imaging the cell andmonitoring the progression of a disease related to B7-H4 expression onthe surface of the cell. In another embodiment the cell is a tumor cellor a tumor-associated macrophage (TAM). In one embodiment, TAMs inhibitT cell proliferation and activation in a B7H4-dependent manner.

In one embodiment, the invention provides a method of restoring T-cellreplication associated with an anti-tumor immune response in a subjectfollowing tumor associated macrophage (TAM)-mediated immune suppressionin the subject, the method comprising the step of administering to thesubject a composition comprising an isolated anti-B7-H4 antibody orfragment thereof, wherein the antibody or fragment thereof is encoded byan amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO: 4.

In one embodiment, the invention provides a method of monitoring a tumorcell frequency in a subject having a tumor growth, the method comprisingthe step of a) obtaining a biological sample from the subject, b)contacting the biological sample with a bioconjugate compositioncomprising the anti-B7-H4-antibody or fragment thereof operably linkedto a labeling agent, and c) monitoring the progression of cell surfaceexpression of B7-H4 in a biological sample, wherein the antibody orfragment thereof is encoded by an amino acid sequence comprising SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In another embodiment, the labeling agent provided herein is ananoparticle quantum dot, a fluorophore, a cyanide, aradioactively-labeled peptide, a magnetic nanoparticle, a chromophore,or a localization marker. In another embodiment, the labeling is carriedout in vivo and enables the imaging of the cell.

In another embodiment, imaging of cells includes any type of imagingtechnique known in the art including in vivo and in vitro imaging. Inanother embodiment, the imaging is optical imaging, fluorescenceimaging, scattering imaging, time-lapse imaging, live imaging,colorimetric imaging, electron microscopy imaging, magnetic resonanceimaging, or a combination thereof.

In another embodiment, the invention relates to a method of effecting atherapeutic T-cell mediated anti-tumor immune response in a subjecthaving an anti-tumor immune response suppression, wherein the immunesuppression is mediated by a B7-H4-expressing cell in the subject, themethod comprising the step of administering to the subject a compositioncomprising a recombinant T-cell specific for the B7-H4-expressing cell,wherein the recombinant T-cell comprises a chimeric antigen receptorcomprising a) an ectodomain comprising an anti-B7-H4 antibody orfragment thereof, b) a transmembrane domain, and c) a T-cell receptorendodomain, wherein the antibody or fragment thereof is encoded by theamino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,or SEQ ID NO: 4.

CAR Composition

The present invention encompasses a recombinant DNA construct comprisingsequences of an antibody of the invention that binds specifically tohuman B7-H4, wherein the sequence of the antibody or a fragment thereofis operably linked to the nucleic acid sequence of an intracellulardomain. The intracellular domain or otherwise the cytoplasmic domaincomprises, a costimulatory signaling region and/or a zeta chain portion.The costimulatory signaling region refers to a portion of the CARcomprising the intracellular domain of a costimulatory molecule.Costimulatory molecules are cell surface molecules other than antigensreceptors or their ligands that are required for an efficient responseof lymphocytes to antigen.

The present invention encompasses a recombinant DNA construct comprisingsequences of a fully human CAR, wherein the sequence comprises thenucleic acid sequence of a B7-H4 binding domain operably linked to thenucleic acid sequence of an intracellular domain. An exemplaryintracellular domain that can be used in the CAR includes but is notlimited to the intracellular domain of CD3-zeta, CD28, 4-1BB, and thelike. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, and the like.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

Antigen Binding Moiety

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding moiety. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR-mediated T-cell response can be directed toan antigen of interest by way of engineering a desired antigen into theCAR. In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin's lymphoma, Hodgkins lymphoma, leukemias, uterine cancer,cervical cancer, bladder cancer, kidney cancer and adenocarcinomas suchas breast cancer, prostate cancer, ovarian cancer, pancreatic cancer,and the like.

In one embodiment, the tumor antigen of the present invention comprisesone or more antigenic cancer epitopes immunologically recognized bytumor infiltrating lymphocytes (TIL) derived from a cancer tumor of amammal.

In a preferred embodiment, the antigen binding moiety portion of the CARtargets B7-H4, preferably human B7-H4.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to monoclonal antibodies, polyclonalantibodies, synthetic antibodies, human antibodies, humanizedantibodies, and fragments thereof. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise a human antibody or a fragment thereof. Thus, in oneembodiment, the antigen biding domain portion comprises a human antibodyor a fragment thereof.

For in vivo use of antibodies in humans, it may be preferable to usehuman antibodies. Completely human antibodies are particularly desirablefor therapeutic treatment of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods using antibody libraries derived from human immunoglobulinsequences, including improvements to these techniques. See, also, U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO91/10741; each of which is incorporated herein by reference in itsentirety. A human antibody can also be an antibody wherein the heavy andlight chains are encoded by a nucleotide sequence derived from one ormore sources of human DNA.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Anti-B7-H4 antibodies directed against thehuman B7-H4 antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies, including, butnot limited to, IgG1 (gamma 1) and IgG3. For an overview of thistechnology for producing human antibodies, see, Lonberg and Huszar (Int.Rev. Immunol., 13:65-93 (1995)). For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; and 5,939,598, each of which is incorporated byreference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above. For a specificdiscussion of transfer of a human germ-line immunoglobulin gene array ingerm-line mutant mice that will result in the production of humanantibodies upon antigen challenge see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993);and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al., Nature,348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of unimmunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905,each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Alternatively, in some embodiments, a non-human antibody is humanized,where specific sequences or regions of the antibody are modified toincrease similarity to an antibody naturally produced in a human. In oneembodiment, the antigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886,International Publication No. WO 9317105, Tan et al., J. Immunol.,169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000),Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem.,272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904(1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995),Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene,150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73(1994), each of which is incorporated herein in its entirety byreference. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well-known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some framework(FR) residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

In some instances, a human scFv may also be derived from a yeast displaylibrary.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

In some embodiments, the antibody is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen, is achieved. In general, the CDR residues are directlyand most substantially involved in influencing antigen binding.

A humanized antibody retains a similar antigenic specificity as theoriginal antibody, i.e., in the present invention, the ability to bindhuman B7-H4. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody for human B7-H4may be increased using methods of “directed evolution,” as described byWu et al., J. Mol. Biol., 294:151 (1999), the contents of which areincorporated herein by reference herein in their entirety.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, theCAR is designed to comprise a transmembrane domain that is fused to theextracellular domain of the CAR. In one embodiment, the transmembranedomain that naturally is associated with one of the domains in the CARis used. In some instances, the transmembrane domain can be selected ormodified by amino acid substitution to avoid binding of such domains tothe transmembrane domains of the same or different surface membraneproteins to minimize interactions with other members of the receptorcomplex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someinstances, a variety of human hinges can be employed as well includingthe human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3-zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR is designedto comprise the CD3-zeta signaling domain by itself or combined with anyother desired cytoplasmic domain(s) useful in the context of the CAR ofthe invention. For example, the cytoplasmic domain of the CAR cancomprise a CD3 zeta chain portion and a costimulatory signaling region.The costimulatory signaling region refers to a portion of the CARcomprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with CD28 and 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB.

Vectors

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-3 (3^(rd) ed., Cold Spring Harbor Press, NY 2001), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the hemoglobin promoter,and the creatine kinase promoter. Further, the invention should not belimited to the use of constitutive promoters. Inducible promoters arealso contemplated as part of the invention. The use of an induciblepromoter provides a molecular switch capable of turning on expression ofthe polynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL volumes 1-3 (4th ed., Cold Spring HarborPress, N Y 2012).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Sources of T Cells

Prior to expansion and genetic modification, a source of T cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable in the art, may be used. In certain embodiments of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutionwith or without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

T cells are activated and expanded generally using methods as described,for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α. or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

In one embodiment, the invention pertains to a method of inhibitinggrowth of a B7-H4-expressing tumor cell, comprising contacting the tumorcell with at least one antibody or a fragment thereof of the inventionsuch that growth of the tumor cell is inhibited.

In one embodiment, the invention pertains to a method of inhibitinggrowth of a B7-H4-expressing tumor cell, comprising contacting the tumorcell with an anti-B7-H4 CAR T cell of the present invention such thatgrowth of the tumor cell is inhibited.

In another aspect, the invention pertains to a method of treating cancerin a subject. The method comprises administering to the subject anantibody or a fragment of the invention or an anti-B7-H4 CAR T cell ofthe present invention such that the cancer is treated in the subject.Particularly preferred cancers for treatment are hepatocellularcarcinomas, pancreatic cancers, ovarian cancers, stomach cancers, lungcancers and endometrial cancers. In still other embodiments, the cancerto be treated is selected from the group consisting of hepatocellularcarcinomas, papillary serous ovarian adenocarcinomas, clear cell ovariancarcinomas, mixed Mullerian ovarian carcinomas, endometroid mucinousovarian carcinomas, pancreatic adenocarcinomas, ductal pancreaticadenocarcinomas, uterine serous carcinomas, lung adenocarcinomas,extrahepatic bile duct carcinomas, gastric adenocarcinomas, esophagealadenocarcinomas, colorectal adenocarcinomas and breast adenocarcinomas.

The present invention includes a type of cellular therapy where T cellsare genetically modified to express a chimeric antigen receptor (CAR)and the CAR T cell is infused to a recipient in need thereof. Theinfused cell is able to kill tumor cells in the recipient. Unlikeantibody therapies, CAR-modified T cells are able to replicate in vivoresulting in long-term persistence that can lead to sustained tumorcontrol. In various embodiments, the T cells administered to thepatient, or their progeny, persist in the patient for at least fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, thirteen months,fourteen month, fifteen months, sixteen months, seventeen months,eighteen months, nineteen months, twenty months, twenty-one months,twenty-two months, twenty-three months, two years, three years, fouryears, or five years after administration of the T cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In another embodiment, the fully-human CARtransduced T cells exhibit specific proinflammatory cytokine secretionand potent cytolytic activity in response to human cancer cellsexpressing the B7-H4, resist soluble B7-H4 inhibition, mediate bystanderkilling and mediate regression of an established human tumor. Forexample, antigen-less tumor cells within a heterogeneous field ofB7-H4-expressing tumor may be susceptible to indirect destruction byB7-H4-redirected T cells that has previously reacted against adjacentantigen-positive cancer cells.

The fully-human CAR-modified T cells of the invention may be a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of diseases, disordersand conditions associated with dysregulated expression of B7-H4. Incertain embodiments, the cells of the invention are used in thetreatment of patients at risk for developing diseases, disorders andconditions associated with dysregulated expression of B7-H4. Thus, thepresent invention provides methods for the treatment or prevention ofdiseases, disorders and conditions associated with dysregulatedexpression of B7-H4 comprising administering to a subject in needthereof, a therapeutically effective amount of the fully humanCAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol, may select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patienttransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i. v.)injection, or intraperitoneally. In one embodiment, the T cellcompositions of the present invention are administered to a patient byintradermal or subcutaneous injection. In another embodiment, the T cellcompositions of the present invention are preferably administered byi.v. injection. The compositions of T cells may be injected directlyinto a tumor, lymph node, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993). In a further embodiment, the cell compositions of the presentinvention are administered to a patient in conjunction with (e.g.,before, simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Anti-B7-H4 Antibodies

The following experiments were designed to isolate and characterizeantibodies that bind to B7-H4. These antibodies are optimal fordevelopment for diagnostic and in vivo therapeutic applications.

The materials and methods employed in these experiments are nowdescribed.

Antibodies

Yeast-display scFv expression was detected with anti-cmyc mousemonoclonal antibody (mAb), 9E10 and Alexa-488 F(ab′)2 fragment of goatanti-mouse IgG (H+L) (488 anti-IgG) or PE-Cy7 goat F(ab′)2 anti-mouseIgG(H+L) (PE-Cu5 anti-IgG). Biotinylated antigen binding to yeastdisplay scFv was detected with goat anti-biotin-FITC or streptavidin-PE.ScFv binding to cell lines was detected with APC-conjugated anti-V5mouse mAb and scFv binding to plastic-immobilized antigen was detectedby HRP-conjugated mouse anti-V5 mAb. Biobody binding to B7-H4-expressercells was detected with APC-labeled streptavidin.

Identification of Anti-B7-H4 scFv

The yeast-display scFv library derived from ovarian cancer patients wasfirst screened by magnetic and flow sorting for anti-B7-H4 scFv usingprogressively decreasing concentrations of human biotinylated B7-H4recombinant protein (rhB7-H4). Briefly, the library was enrichedmagnetically 3 times for scFv that bound to 1 ug/ml biotinylatedrhB7-H4, and two times flow sorted with 1 ug/ml and 0.5 ug/mlbiotinylated rhB7-H4. Selected yeast-display scFv were flow sorted forc-myc/B7-H4 double positive clones. DNA plasmids were extracted fromyeast display scFv fragments were amplified using primers allowinghomologous recombination with the yeast secretion vector p416-BCCP. Theprimers used were: Forward shuffling primer:5′-ggttctggtggtggaggttctggtggtggtggatctg-3′(SEQ ID NO: 11); Reverseshuffling primer:5′-gagaccgaggagagggttagggataggcttaccgtcgaccaagtcttcttcagaaataagctt-3′(SEQ ID NO: 12). ScFv fragments and linearized p416-BCCP wereco-transfected into YVH10 by chemical transformation. Soluble scFvscreening for specific binding to biotinylated B7-H4 was performed bycapture ELISA using high through put purified yeast supernatants of 100random transformants immobilized in amino plates. Binding to serialdilutions of biotinylated rB7-H4 or control protein was detected usingstreptavidin-HRP. Colorimetric signals were developed with TMB substratesolution quenched with sulfuric acid and read at 450 nm on a Fluoroskandeviations Sequencing of scFv clones 26 and 56 was identified 2 uniqueclones that were then produced and Ni purified.

Measurement of scFv Affinity by ELISA

To assess scFvs affinity, ELISA plates were coated with rhB7-H4 attwo-fold decreasing concentrations from 0.4 to 0.05 μg/ml, incarbonate-bicarbonate buffer. After blocking with PBSM, wells wereincubated with ten-fold serial dilutions of scFv, starting from 1 μM.ScFv binding to immobilized proteins was detected with HRP anti-V5.Colorimetric signals were then developed.

Flow Cytometry Analysis

Analysis of scFv expression by yeast was performed. Briefly, binding ofanti-B7-H4 scFv and biobodies to B7-H4-expresser macrophages and tumorcells was evaluated Anti-B7-H4 scFv were preincubated for 30 min at RTwith APC-anti-V5 at a ratio 1/1 and anti-B7-H4 biobodies werepreincubated with FITC-labeled streptavidin beads. A non-relevant scFvor biobody was used as a negative control for binding.

Orthotopic Mouse Model of Ovarian Cancer

MOV1 mouse ovarian cancer cell line is derived from an ovarian cancerthat spontaneously arises in female transgenic mice that express thetransforming region of SV40 under control of the Mullerian inhibitorysubstance type II receptor gene promoter (Tg-MISIIR-Tag). MOV1 cell lineexpresses SV40 antigen. To emulate ovarian cancer mouse ovarian cancercells, MOV1 were orthotopically injected in the ovarian bursa ofNOD-Scid-γ null (NSG) mice. Four month-old multiparous females areanesthetized according to the protocol approved by the University ofPennsylvania Institutional Animal Care and Use Committee (IACUC). Adorsolateral incision on left caudal portion of the animal dorsum wasmade. The retroperitoneum is dissected to expose the left ovary usingthe forceps to grasp, retract, position, and secure the organ forinjection. Five million MOV1 cells are injected in the ovarian bursa ina volume of 20 μl of PBS using an insulin syringe. Retroperitonealincisions are closed, animals are administered antibiotics and fluids,and tumor growth is monitored by in vivo imaging.

Analysis of In Vivo Distribution of Anti-B7-H4 Biobodies by ConfocalMicroscopy

Anti-B7-H4 biobody-26 of high affinity is injected intravenously (IV) 3weeks after tumor cell implantation. As control for the EnhancedPermeability and Retention (EPR) effect, the anti-B7-H4 biobodies of lowaffinity is used. Spleen, liver, kidney and ovaries are harvested 24 or48 h after biobody injection and preserved in frozen tissue matrix OCTcompound. Slides of 5μ thickness are cut from frozen sections, air dried1 h at RT and fixed by immersion in cold 100% acetone 5 min. After 2washes in PBS, slides are blocked for endogenous biotin by pre-treatmentwith avidin/biotin blocking solution (avidin-skim milk 0.001% in PBS).Anti-B7-H4 biobody binding are detected with rhodamine-conjugatedstreptavidin. MOV1 tumor cells derived from a Tg-MISIIR-TAg tumorexpress SV40 and thus could be detected with anti-SV40 Tag antibody (2μg/ml) for 30 min at RT, followed by 1 μg/ml Alexa-488 goat anti-mouseIgG1κ for 30 min at RT. Slides are incubated with 1/2000 diluted DAPIfor 30 min at RT to visualize the nuclei. Fluorescent signals areacquired by confocal analysis at 63× magnification.

The results of the experiments are now described.

Tumor Cells and Monocytes from Ascites and Solid Tumors of OvarianCancer Patients Express B7-H4 at the Cell Surfaces

B7-H4 cell surface expression has been shown for tumor-associatedmacrophages but not in ovarian cancer cells. Without being bound by anyparticular theory, it is believed that tumor cell surface expression ofB7-H4 permits targeting and suggests the existence of alternate immuneinhibitory functions of ovarian tumor cells.

Analysis of B7-H4 surface expression on tumor cells using flow cytometryrevealed that these tumor cells indeed expressed B7-H4 on the cellsurface (FIGS. 1A-1B). Hence, it was confirmed that B7-H4 is expressedat the cell surface of fresh ovarian cancer cells (FIG. 1A) andmonocytes (FIG. 1B).

Macrophages Up-Regulate B7-H4 Expression after Co-Culture with OvarianCancer Cells

Analysis of B7-H4 surface expression on monocytes from ascites and solidtumors from ovarian cancer patients using flow cytometry revealed thatthese tumor cells indeed expressed B7-H4 on the cell surface (FIGS.2A-2D). Hence, it was confirmed that B7-H4 is expressed at the cellsurface of macrophages freshly isolated from ovarian ascites or solidtumors (FIG. 2A), as well as of macrophages maturated in presence of IL4and IL10 (FIG. 2B). In addition, it was demonstrated that co-culturewith B7-H4 expresser OvCar3 tumor cell line also up-regulates theexpression of B7-H4 (FIG. 2B).

Macrophages Strongly Up-Regulate B7-H4 Expression after Co-Culture withB7-H4+Ovarian Cancer Cell Line

In vitro model system of cell co-culture in transwell was carried out,allowing chemical exchanges between human monocyte-derived macrophagesand human ovarian cancer cell line Ovcar3. Co-culture of macrophageswith Ovcar3 polarized macrophages towards TAM phenotype.

Transwell co-culture of macrophages with tumor cells enables a superiorup regulation of B7-H4 on macrophages compared to cytokine (IL4/IL10)stimulation. This model system allows for studying tumor cell inductionof B7-H4 on macrophages.

Isolation of Anti-B7-H4 Recombinant Antibodies (scFvs) from aYeast-Display scFv Library

B7-H4 extracellular domain was cloned from macrophages co-cultured withovcar3 and expressed in mammalian cells. ScFvs against B7-H4 wereisolated by screening a yeast-display scFv library. ScFvs were convertedinto soluble form and specific binding was validated on recombinantB7-H4 protein (FIG. 3A) and on macrophages (FIG. 3B).

These results demonstrate that the novel anti-B7-H4 biobodies in complexwith streptavidin iron oxide FITC labeled beads are more sensitive thancommercially available anti-B7-H4 mAb. In addition, it demonstrates thatthe ovcar5 cell line expresses B7-H4 since utilizing the novel scFv'sB7-H4 can be detected even at low expression levels (ovcar5) that cannotbe detected with commercial mAb anti-B7-H4.

The results further demonstrate that an established ovarian cancer cellline, OvCar3, expresses B7-H4 at the cell surface; and a sub-populationof OvCar5 cell line also expresses B7-H4.

B7-H4 Expression on Macrophages and Tumor Cell's Frequency

After measuring the expression of B7-H4 on macrophages and the tumorcell's frequency, it was found that the two were positively correlated,in that B7-H4 expression on monocytes was correlated with the percentageof tumor cells in ovarian cancer samples.

Anti-B7-H4-Mediated Blockage of B7-H4-Mediated T Cell Suppression byMacrophages Promotes T Cell Proliferation

It was further demonstrated that anti-B7-H4 clone scFv #26 could blockB7-H4-mediated T cell suppression by macrophages and promote anti-tumorT cell proliferation (FIGS. 4A-4E).

Therefore, scFv 26, can unexpectedly restore T cell proliferationagainst tumor cells in presence of macrophages and hence can be a usefultherapeutic composition against these tumor cells.

Example 2 Novel Human Anti-B7-H4 Recombinant Antibodies OvercomeB7-H4-Mediated T-Cell Inhibition and Potentiate T Cell Anti-TumorResponses

B7-H4 (B7x/B7s), one of the most recently identified members of B7superfamily, serves as an inhibitory modulator of T-cell responses.B7-H4 is expressed by various human cancers and B7-H4 expression bymacrophages has been significantly correlated with advanced stages ofovarian cancer and with high numbers of tumor-infiltrating T regulatorycells. B7-H4 expressed at the surface of tumor-associated macrophages(TAMs) or surrogate APCs negatively regulates T cell activation,possibly through interaction with a putative ligand, and B7-H4 blockingby antisense oligonucleotides inhibited tumor-associated macrophagessuppression and enabled anti-tumor T cells in vitro and in vivo.However, to this date, B7-H4 cell surface expression has been poorlyunderstood.

The following experiments were designed to study cell surface expressionof B7-H4 in samples from ovarian cancer ascites and solid tumors, aswell as in human ovarian cancer cell lines after passage and short termculture. B7-H4 was expressed at the cell surface of tumor-infiltratingmonocyte/macrophages and of freshly harvested Epcam⁺ tumor cells fromcancer patients or from xenograft mouse model, but the B7-H4 expressionon tumor cells was downregulated after short term in vitro culture.

Four anti-B7-H4 recombinant antibodies (scFvs) were isolated bydifferential screenings of a yeast-display scFv library derived fromhuman B lymphocytes from ovarian cancer patients. Three out of fouranti-B7-H4 scFvs could reverse T-cell inhibition mediated by a B7-H4recombinant protein, as demonstrated by IFN-γ secretion, CD69 expressionand T cell proliferation in response to anti-CD3 stimulation.Furthermore, antigen-specific T cell responses were inhibited by B7-H4⁺antigen-loaded APCs or B7-H4⁺ tumor cells (presentation in cis), and byB7-H4⁺ APCs in presence of tumor cells (presentation in trans). Thepresence of anti-B7-H4 scFv 3#68 in antigen-specific T cell co-cultureswith B7-H4⁺ antigen-loaded APCs, B7-H4⁺ tumor cells, or B7-H4⁺ TAMs andtumor cells, fully restored B7-H4-dependent inhibition. These datapresented herein demonstrate that B7-H4 cell surface expression isinducible in vivo by the tumor microenvironment and show that blockingB7-H4 with an antibody restores anti-tumor T cell responses in vitro.Anti-B7-H4 scFvs of the invention can be used for immunotherapy againstsolid tumors.

The materials and methods employed in these experiments are nowdescribed.

Human Samples

Ascites and solid tumors samples from ovarian cancer patients wereobtained from the Ovarian Cancer Research Center's patient samplerepository of the University of Pennsylvania. Purified T cells andmonocytes from healthy donors were obtained from the Human ImmunologyCore of the University of Pennsylvania.

Human Ovarian Cancer Cell Lines

A1847, OVCAR3, C30, T2, 624, MDA231, OVCAR5 were obtained from ATCC. M2macrophages are generated as previously described (Dangaj et al., 2011,PLoS One 6(12):e28386). EBV-B cells were kindly provided by Dr. RajSomasundaram (Wistar Institute, Philadelphia, Pa.).

Xenograft Model of Ovarian Cancer

Balb/c nude mice were obtained from Charles River Laboratories.

Mice of 6-8 weeks were injected intraperitoneally with 3×10⁶ of OVCAR5ovarian cancer cell line. Mice were sacrificed at 6-9 weeks after tumorimplantation. Ascites and solid tumor samples were collected andanalyzed by flow cytometry.

Generation of a Yeast-Display Recombinant Antibodies (scFv) LibraryDerived from B Cells of Ovarian Cancer Patient Ascites

B cells used for VL/VH amplification were isolated from ascites of fiveovarian cancer patients (stages III or IV) after Ficoll gradient andpurification using CD19 magnetic beads (Miltenyi). Total RNA wasextracted (1.25 mg) and twenty-two μg of mRNA (1.7%) were isolated (mRNApurification kit, Qiagen), consistent with the fact that mRNA normallyaccounts for 1-5% of total RNA. Fifty reverse transcription reactionswere performed to permit the PCR amplification of the VH and VL genefragments. A set of primers was designed to amplify human subfamilies ofVH and VL gene fragments based on the MIGT data base (Sblattero andBradbury, 1998, Immunotechnology 3(4):271-8). Annealing sequences wereadded to primers amplifying VH and VL fragments to enable gap repairwith pAGA2 vector and the generation of a long-linker between VL and VH(GGSSRSSSSGGGGSGGGG; SEQ ID NO: 13) (Andris-Widhopf et al., 2011, ColdSpring Harb Protoc, 2011(9)). The long-linker DNA sequence was modifiedto encode yeast-optimized codons(5′-ggtggttcctctagatcttcctcctctggtggcggtgg ctcgggcggtggtggg-3′; SEQ IDNO: 14).

The following annealing sequences were added to the primers:5′-ggtggtggaggttctggtggtggtggatctgtc-3′; SEQ ID NO: 15 to forward VLprimers (annealing with 5′ end of linearized Nhe I-Xho I pAGA2 vector);5′-cgctgccaccgccgccgctggaacttgacctagaggatccgcc-3′; SEQ ID NO: 16 toreverse VL primers (annealing with long-linker);5′-ctaggtcaagttccagcggcggcggtggcagcggaggcg gcggt-3′; SEQ ID NO: 17 toforward VH primers (annealing with long-linker); and3′-gtcttcttcagaaataagcttttgttcggatccctcgaa-5′; SEQ ID NO: 18 to reverseVH primers (annealing with 3′ end of linearized Nhe I-Xho I pAGA2vector). PCR amplification were performed using a hot start of 5 min at94° C., followed by 25 cycles of denaturation at 94° C. for 1 min,annealing at 55° C. for 1 min and extension at 72° C. for 1 min, with afinal step of extension of 7 min at 72° C. The pAGA2 yeast displayvector was linearized by Nhe I and Xho I. Linearized vector and PCRproducts were separated by electrophoresis, purified using a gelextraction kit (Invitrogen, Carlsbad, Calif.), and transfected intoEBY100 yeast cells at a 1/3/3 ratio (FIG. 6A). Gap repair efficiency wasevaluated by sequencing of fifty clones, as previously described (Zhaoet al., 2011, J Immunol Methods 363(2):221-32).

Cloning of Recombinant B7-H4, Protein Expression and Purification

The extracellular domain of B7-H4 (IgC+IgV) was amplified from cDNA ofhuman macrophages after in vitro tumor-polarization (Dangaj et al.,2011, PLoS One 6(12):e28386), using B7-H4 forward primer5′-ggttctggtggtggaggttctggtggtggtggatctgagtttggtatttcagggagacactccatca-3′; SEQ ID NO: 19 andB7-H4 reverse primer5′-agaccgaggagagggttagggataggcttaccgtcgacagaagcctttgagtttagcagctgtag-3′; SEQ ID NO: 20. B7-H4 cDNA was verified by sequencing andcloned into a mammalian expression vector (pTT28, kind gift from YvesDurocher, National Research Council of Canada) (FIG. 2B) for mammalianexpression fused to 6λHIS Tag in 293-F mammalian cells. RecombinantB7-H4 (rB7-H4) was purified with Nickel sepharose beads (Sigma) anddetectable by Western Blot as a 75 KDa fragment with an anti-B7-H4polyclonal antibody (FIG. 11C).

Construction of pELNS-B7-H4 Lentivirus

For the production of cDNA encoding full B7-H4, RNA was isolated fromOVCAR-3 ovarian tumor cells and reverse transcript with the kit“ready-to-go you-prime First-Strand Beads” (GE Healthcare, Piscataway,N.Y., USA). Resulting cDNA was used as template for PCR amplification ofB7-H4 cDNA fragment of 795-bp with the primers B7-H4-F5′-ACGCTCTAGAATGGCTTCCCTGGGGCAGATC CTCT-3′; SEQ ID NO: 21 and B7-H4-R:5′-ACGCGTCGACTTATTTTAGCATCA GGTAAGGGCTG-3′; SEQ ID NO: 22. The resultingPCR products contained an XbaI site (B7-H4-F) and a SalI site (B7-H4-R)was and were digested for cloning into a third generationself-inactivating lentiviral expression vector (pELNS, (Lanitis et al.,2012, Mol Ther 20(3):633-43)) in which the transgene expression isdriven by the EF-1a promoter, to obtain pELNS-B7-H4.

Recombinant Lentivirus Production

High-titer replication-defective lentiviral vectors were produced andconcentrated as previously described (Lanitis et al., 2012, Mol Ther20(3):633-43). 293T human embryonic kidney cells were seeded at 10×10⁶per T-150 tissue culture flask 24 h before transfection. All plasmid DNAwere purified using the QIAGEN Endo-free Maxi prep kit. Cells weretransfected with 7 μg pVSV-G (VSV glycoprotein expression plasmid), 18μg of pRSV.REV (Rev expression plasmid), 18 μg of pMDLg/p.RRE (Gag/Polexpression plasmid), and 15 μg of pELNS transfer plasmid using ExpressInn (Open Biosytems). Viral supernatant was harvested at 24 and 48 hpost-transfection. Viral particles were concentrated byultracentrifugation for 3 h at 25,000 rpm with a Beckman SW28 rotor(Beckman Coulter, Fullerton, Calif.) and resuspended in 4 ml of RPMIfull medium.

Lentiviral Transduction of Cancer Cell Lines and T2 Cells withpELNS-B7-H4

For the transduction of the cancer cell lines C30, MDA 231 and 624 withpELNS-B7-H4 lentiviral particles 1.5×10⁵ tumor cells were seeded in asix-well plate one day prior their transduction. Next day, the mediumwas replaced with 1 ml of lentivirus when the cells reached a confluenceof about 30%. Medium was replaced twenty four hours after transductionwith fresh RPMI medium (C30 and 624) or DMEM medium (MDA231). For thetransduction of T2 cells, 1 ml of lentivirus was applied to 3×10⁵ cellsin a 24 well plate. The expression of B7-H4 was assessed at day 5 posttransduction.

Production of Retroviral Particles by Transient Transfection of 293 GPCells

The HER-2 TCR and the MART-1 TCR (DF5a) (Johnson et al., 2006, J Immunol177(9):6548-59) used in this study were in the pMSGV1 vector backbonewhich is a derivative of the vector pMSGV (murine stem cell virus(MSCV)-based splice-gag vector) and utilizes a MSCV long terminal repeat(LTR) (Zhao et al., 2005, J Immunol 174(7):4415-23). To produceretroviral supernatants, 1×10⁶ of 293-GP cells (transient viral producercells) in a 6-well plate were co-transfected with 1.5 μg of retroviralvector DNA from each of the constructs and 0.5 μg of envelope DNA(RD114) using the Lipofectamine 2000 reagent (Invitrogen) and Optimemmedium (BD Biosciences). Media was changed to DMEM with 10% FBS after 18h, and viral supernatants were harvested at the 48-h time point. Thesesupernatants were then used to transduce T cells for expression of a TCRthat targets either HER-2- or MART-1-derived peptide.

Isolation of Anti-B7-H4 scFvs from Yeast Display scFv Library

Anti-B7-H4 scFvs were first selected by magnetic and flow sorting usingrB7-H4 vs. control protein, as previously described (Dangaj et al.,2011, PLoS One 6(12):e28386; Zhao et al., 2011, J Immunol Methods363(2):221-32). In addition, the selected subpopulation of yeast-displayscFvs were further selected by panning using a protocol derived fromWang et al. (Wang et al., 2007, Nat Methods 4(2):143-5) with thefollowing specifications: C30 ovarian cancer cell line was transducedwith pELNS-B7-H4 or with pELNS-GFP as negative control, and grown inmonolayer to 90% confluence on poly-L-Lysine-coated dishes. Yeast wereinduced to express scFv, washed with PBE and depleted for non-specificbinding by 2 incubations with GFP⁺ C30 cells at a ratio of 30-60:1(yeast:cells) for 30 min at RT with gentle rotation to prevent clumping(1-2 speed). Unbound yeast were then harvested and incubated withplastic-immobilized B7-H4⁺ C30 cells for 30 min at RT with gentlerotation. Plates were washed twice with PBS for 5 min at RT with gentlerotation and examined under microscope. Yeast clusters that were stillbinding to cells were harvested and grown on the cell plate O/N andtransferred to new flask for further amplification. Yeast panning wasrepeated 4 times. Yeast displayed scFvs were converted into solubleforms as described previously (Dangaj et al., 2011, PLoS One6(12):e28386; Zhao et al., 2011, J Immunol Methods 363(2):221-32).

Human T Cell Transduction

Primary human T cells were purchased from the Human Immunology Core atUniversity of Pennsylvania and were isolated from healthy volunteerdonors following leukapheresis by negative selection. All specimens werecollected under a University Institutional Review Board-approvedprotocol, and written informed consent was obtained from each donor. Tcells were plated at a concentration of 1×10⁶/ml in 24-well plates(Costar) in complete media (RPMI 1640 supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μm/mlstreptomycin sulfate, 10 mM HEPES), and stimulated with anti-CD3 andanti-CD28 mAbs coated beads as described by manufacturer (Invitrogen)(Levine et al., 1997, J Immunol, 159(12):5921-30) for 18-24 h prior totransduction. Non-tissue culture-treated 12-well plates (BectonDickinson Labware, Franklin Lakes, N.J.) were treated with 25 ug/ml ofrecombinant retronectin fragment at 4° C. as directed by themanufacturer (RetroNectin, Takara, Otsu, Japan). After an overnightincubation, the retronectin was removed and the plate was blocked with2% BSA in PBS at RT for 30 minutes. The retroviral vector supernatant(2-3 ml) was then applied by centrifugation (2000×g for 2 hours), andafter discarding the supernatant 5×10⁵ of stimulated T cells were addedto each well in a final volume of 1 ml RMPI growth medium per well.Plates were centrifuged for 10 min at 1000×g and incubated overnight.The transduction process was repeated the following day. Aftertransduction, the cells were grown in the RMPI with 10% FBS and humanrecombinant interleukin-2 (Novartis) was added every other day to a 100IU/ml final concentration. Cell density of 0.5-1×10⁶ cells/ml wasmaintained. Expression of the exogenous HER-2 and MART-1 TCR wasvalidated 5 days after transduction using APC-conjugated MHC-peptidetetramers (Becton Dickinson, San Jose, Calif.) with specificities forHLA-A2-HER2369 and HLA-A2-MART-1.

T Cell Activation

B7-H4 inhibition of T cell activation and proliferation was performedusing plate-immobilized recombinant B7-H4. A day prior to T cellactivation, antibodies (anti-CD3 mAb (clone OKT3) and/or anti-CD28(eBiosciences) were plastic-immobilized at 5 μg/ml and 2 μg/mlrespectively, in 100 μl/well of bicarbonate buffer on flat 96-welltissue culture plates, overnight. The antibody solution was removed theday of T cell activation, and 10 μg/ml of rB7-H4 protein was coated in100 μl/well of bicarbonate buffer for 2 hrs at 37° C. A non relevantrecombinant protein (folate receptor alpha) was used as control protein.In the meantime, T cells were labelled with 3 μM of CFSE, as instructedby manufacturer (Invitrogen). Plates were then washed two times with PBSand labelled T cells were cultured at 1×10⁵ in 150 μl/well in thepresence of anti-B7-H4 scFvs added at day 0 at a concentration of 2.5μg/ml. T cell responses were analyzed five days after activation. Assayswere performed in triplicates.

T Cell Co-Culture with T2 APCs

Wild type, GFP- or B7-H4-transduced T2 APCs were resuspended at10×10⁶/ml and loaded with HER-2 or MART-1 peptides at various peptideconcentrations at 37° C. for 2 hrs. Co-culture of peptide-loaded APCswith HER-2 or MART-1 TCR specific T cells was performed at ratio of 1:1(1×10⁵T2/1×10⁵T cells) in 200 μl of RPMI media in round bottom 96-welltissue culture plates. Anti-B7-H4 scFvs were added at day 0 at 5 μg/mlof concentration. T cell responses were analyzed two days afterco-culture. Assays were performed in triplicates.

TAM-mediated TCR T cell inhibition assay were performed in 24 welltranswell co-cultures at ratio of 1:1:1 (1×10⁵ T2/1×10⁵ T cells/1×10⁵TAM). TAMs were differentiated and polarized as described previously(Dangaj et al., 2011, PLoS One 6(12):e28386). Briefly M1 polarizedmacrophages were in transwell co-cultured with OVCAR3 ovarian cancerline for 3 days. At day 3 of tumor polarization of macrophages, TCR Tcells and peptide pulsed wild type T2 cells were added in the transwellscontaining or not macrophages. Anti-B7-H4 scFvs were added at day 0 at 5ug/ml concentration. T cell responses were analyzed three days afterco-culture. Assays were performed in triplicates.

T Cell Co-Culture with Tumor Cells

Wild type or B7-H4⁺ 624 melanoma cell line and/or wild type or B7-H4⁺MDA231, were co-cultured with HER-2 or MART-1 TCR specific T cells at aratio of 1:1 (1×10⁵ T2/1×10⁵ T cells) in 200 μl of RPMI media. 5 μg/mlof anti-B7-H4 scFvs were added at day 0 and T cell responses wereanalyzed two days after co-culture. Assays were performed intriplicates.

Flow Cytometry

Flow cytometry was performed as described (Dangaj et al., 2011, PLoS One6(12):e28386). Before labelling cells were incubated with mIgG to blocknon-specific binding of Fcγ Receptors. 7-AAD was used to exclude deadcells.

ELISA

IFN-γ ELISA was performed as indicated by manufacturer (Biolegend).

Western Blotting

Western blotting was performed as described (Dangaj et al., 2011, PLoSOne 6(12):e28386).

Statistics

P values were calculated using One Way Anova and Unpaired T testanalysis.

The results of the experiments are now described.

B7-H4 Expression at the Cell Surface of Tumor-Infiltrating Monocytes andTumor Cells is Up-Regulated In Vivo and Down Regulated by In VitroCulture

Cell surface-targeting requires the presence of membrane-boundmolecules. B7-H4 cell surface expression in established ovarian cancercell lines (n=5) and in ovarian cancer samples (ascites and solidtumors) (n=16) analyzed using flow cytometry. It was observed that cellsurface expression of B7-H4 was limited in established ovarian cancercell lines (1 out of 5, OVCAR3, FIG. 5A), consistently with previousreport (Choi et al., 2003, J Immunol 171(9):4650-4). As positivecontrols of B7-H4 cell surface expression, EBV B cells were used (Quandtet al., 2011, Clin Cancer Res 17(10):3100-11), in vitro differentiatedmacrophages stimulated with IL4 and IL-10, and B7-H4 transduced C30cells (FIG. 5A, as indicated). In contrast, a broad cell surfaceexpression of B7-H4 among 16 solid tumors and ascites from ovariancancer patient samples was observed (FIG. 5B; Tables 1 and 2). MeanB7-H4 expression in CD45⁻ Epcam⁺ tumor cells was of 28%±17.84 in ascites(Table 1) and 9.44%±8.31 in solid tumors (Table 2). Twenty five percentof patients had more than 30% of CD45⁻ Epcam⁺ tumor cells expressingB7-H4, and more than half of the patients expressed B7-H4 on 4 to 15% oftheir tumor cells. B7-H4 expression was also observed in CD45⁻ Epcam⁻cells derived from solid tumors (Table 2). In addition, B7-H4 cellsurface expression on tumor-associated CD45⁺CD14⁺ monocytes wasconfirmed, as reported by Cryczek et al. (Kryczek et al., 2006, J ExpMed 203(4):871-81), and 3 to 40% of CD45⁺CD14⁺ cells expressed bothB7-H4 and CD206/mannose receptor, a marker of mature M2 macrophages andof TAMs (Table 2). To address whether B7-H4 expression in tumor cells isan in vivo inducible effect, a human ovarian cancer cell line withundetectable surface B7-H4 expression, OVCAR5 (FIG. 5A), was used toestablish intraperitoneal tumors in Balb C/nude mice. Nine weeks aftertumor injection, 6 ascites and solid tumors were collected and analyzedfor B7-H4 expression by flow cytometry. B7-H4 cell surface expression inEpcam⁺ tumor cells was upregulated in all freshly harvested OVCAR5tumors (n=6), ranging from 4 to 38% (mean=12.8±5.18) (FIGS. 5C and 5D).However, after a short term culture of OVCAR5 cells from ascites orsolid tumor, B7-H4 cell surface expression was back to undetectablelevels (FIGS. 5C and 5D). These results demonstrated that B7-H4 cellsurface expression in ovarian tumor cells is induced in vivo anddownregulated by short term culture.

TABLE 1 B7H4 expression on tumor-infiltrating monocytes and tumor cellsin human ovarian cancer ascites % CD45⁺ % % CD45⁺ % CD45⁺ CD14⁺ % CD45⁻Specimen % CD45⁺ CD14⁺ CD14⁺ CD206⁺ CD45⁻ Epcam⁺ ID % CD45⁺ CD14⁺ B7H4⁺CD206⁺ B7H4⁺ Epcam⁺ B7H4⁺ 1647 97.90 40.00 40.5 49.00 58.16 1.00 32.001686 98.90 20.30 0.70 58.00 1.72 0.15 13.00 1714 96.50 3.49 1.80 42.002.86 2.10 13.36 1753 95.70 72.80 17.80 55.00 32.73 2.43 14.74 1773 71.7037.40 25.00 75.00 22.67 23.30 38.90 1756 49.00 49.00 17.00 52.00 48.0842.90 57.00 Mean 84.95 37.16 17.13 55.17 27.7 11.98 28.17 St. Dev 20.3923.81 14.92 11.16 23.18 17.51 17.84Surface B7-H4 expression was assessed in human ovarian cancer ascitesusing flow cytometry (n=6); results were expressed in percentage ofstained cells and plotted as indicated.

TABLE 2 B7H4 expression on tumor-infiltrating monocytes and tumor cellsin human ovarian solid tumors % % CD45⁺ % % % CD45⁺ % CD45⁺ CD14⁺ %CD45⁻ % CD45⁻ % CD45⁺ CD14⁺ CD14⁺ CD206⁺ CD45⁻ Epcam⁺ CD45⁻ Epcam⁻ IDCD45⁺ CD14⁺ B7H4⁺ CD206⁺ B7H4⁺ Epcam⁺ B7H4⁺ Epcam⁻ B7H4⁺ 1789 10.5039.40 29.75 58.00 39.65 9.20 7.00 76.00 0.00 1790 24.70 26.00 22.8 36.0027.78 6.86 11.00 66.50 0.00 1746 45.58 21.62 15.00 35.00 29.29 49.7813.00 0.00 — 1751 62.50 21.40 8.40 42.00 20.24 12.30 29.80 22.30 11.751761 66.10 23.20 7.75 42.00 14.52 11.50 9.60 20.30 10.62 1767 86.00 5.261.70 70.00 2.86 0.00 0.00 11.80 3.99 1791 4.66 40.80 13.00 70.00 14.2937.20 5.00 54.60 5.20 1761 51.80 61.20 7.69 45.00 13.33 4.18 12.00 42.504.00 1791 44.00 43.00 10.00 25.00 4.73 35.00 4.00 16.30 31.00 1801 40.0025.00 44.00 32.00 17.20 27.00 3.00 27.00 13.00 Mean 43.58 30.69 16.1045.50 18.39 19.30 9.44 33.73 8.84 St. Dev 25.20 15.59 12.757 15.59 11.3016.74 8.31 25.06 9.60Surface B7-H4 expression was assessed in human solid ovarian tumorsusing flow cytometry (n=10); results were expressed in percentage ofstained cells and plotted as indicated.Generation of a Yeast Display Library Derived from Tumor-Associated BCells from Ovarian Cancer Patients

A novel yeast-display library of recombinant antibodies (scFvs) derivedfrom the variable regions of the heavy and light chains of B cellsisolated from human ovarian cancer ascites (n=10) and PBMCs (n=1) wasconstructed. The insertion of V_(H)-V_(L) fragments encoding the scFv inpAGA2 vector (Zhao et al., 2011, J Immunol Methods 363(2):221-32) wasperformed using homologous recombination in yeast combining V_(H) PCRfragments, V_(L) PCR fragments and linearized pAGA2 vector. V_(H) andV_(L) were linked together by the linker (GGSSRSSSSGGGGSGGGG; SEQ ID NO:13 (Zhang et al., 2012, Mol Ther 20(7):1298-304; Andris-Widhopf et al.,2011, Cold Spring Harb Protoc, 2011(9)). The diversity of the librarywas estimated at 10⁹.

Isolation and Validation by Capture ELISA of Novel Anti-B7-H4 scFvs

The method of soluble scFv isolation is described elsewhere herein andrecapitulated in FIG. 6A. Briefly 3 magnetic and 2 flow sortings of theyeast-display scFv library was performed from which a subpopulation ofyeast display scFvs that bound to soluble recombinant B7-H4 (rB7-H4) wasisolated. The selected subpopulation of yeast-display scFvs was thendirectly transformed in soluble scFvs by homologous recombination inp416-BCCP vector, as previously described (Zhao et al., 2011, J ImmunolMethods 363(2):221-32; Scholler et al., 2006, J Immunol Methods317(1-2):132-43; Bergan et al., 2007, Cancer Lett 255(2):263-74) asreferred to as “protein-based screening strategy.” Alternatively,screening was completed by further enrichment with four rounds ofpanning on C30 cell line transduced to express B7-H4 at the cellsurface, or GFP as negative control. This screening method is referredto as “cell-based screening strategy.” After transformation into solubleforms, isolated scFvs were high-throughput purified from yeastsupernatants (Zhao et al., 2011, J Immunol Methods 363(2):221-32; Berganet al., 2007, Cancer Lett 255(2):263-74), and screened by capture ELISAfor specific binding to 500 ng/ml of rB7-H4 (data not shown). Two scFvs(#26 and #56) issued from the protein-based screening strategy, and 2scFvs (3#54 and 3#68) issued from the cell-based screening strategy wereselected for further analysis. Selected scFv were assayed by captureELISA for binding to serial dilutions of rB7-H4 with similar results;BSA was used as control protein (FIGS. 6B-6C). ScFvs were sequenced andanalyzed for their gerrmline immunoglobulin gene usage of the predictedamino-acid sequence by the Kabat system (Table 3). Immunoglobulin geneusage comparison of protein-based isolated scFv clones #26 and #56demonstrated substantial differences of immunoglobulin gene usage inboth heavy and light chains. ScFv clones 3#54 and 3#68 displayeddifferent immunoglobulin gene usage for heavy chains, but essentiallysame light chains. Clone #26 and 3#68 shared the same IGHV and IGHD geneusage but different IGHJ genes for the heavy chains and different usagefor the light chains. Sequence identifiers for the scFvs are depicted inTable 4:

TABLE 3 Germline immunoglobulin gene usages of the predicted amino-acidsequence of the anti-B7-H4 scFvs B7-H4 scFv Ig Gene HEAVY CHAIN LIGHTCHAIN Usage VH GENE D GENE JH GENE VL GENE JK, L GENE 26 scFv IGHV1-IGHD5- IGHJ6*02 IGKV3- IGKJ2*02 2*02 24*01 (94.1%) 20*01 (97.0%) (92.1%)(91.7%) (94.8%) 56scFv IGHV4- IGHD6- IGHJ3*02 IGLV6- IGLJ3*02 39*0713*01 (100%) 57*01 (91.4%) (98.7%) (100%) (93.8%) 3#54 scFv IGHV4-IGHD6- IGHJ6*02 IGLV1- IGLJ3*02 b*02 19*01 (100%) 47*01 (97.3%) (93.2%)IGHD6- (97.9%) 13*01 (100%) 3#68 scFv IGHV1- IGHD5- IGHJ3*01 IGLV1-IGLJ3*02 2*02 24*01 (100%) 47*01 (94.6%) (92.1%) (91.7%) (98.3%)Kabat analysis of the homology of heavy (H) and light (L) chain variableregions to germline immunoglobulin genes is displayed for eachanti-B7-H4 scFv clone.

TABLE 4 Sequence Identifiers for anti-B7-H4 antibodies SEQ ID NO: #IDENTITY SEQ ID NO: 1 anti-B7H4 scFv clone 56 (amino acid) SEQ ID NO: 2anti-B7H4 scFv clone 26 (amino acid) SEQ ID NO: 3 anti-B7H4 scFv clone54 (amino acid) SEQ ID NO: 4 anti-B7H4 scFv clone 68 (amino acid) SEQ IDNO: 5 anti-B7H4 scFv clone 56 (nucleic acid) SEQ ID NO: 6 anti-B7H4 scFvclone 26 (nucleic acid) SEQ ID NO: 7 anti-B7H4 scFv clone 54 (nucleicacid) SEQ ID NO: 8 anti-B7H4 scFv clone 68 (nucleic acid)Recombinant B7-H4 (rB7-H4) Protein Inhibits T Cell Polyclonal Activationand Anti-B7-H4 scFvs Blocks rB7-H4-Dependant T Cell Inhibition

Normal donor T cells were stimulated with anti-CD3 and/or anti-CD28immobilized antibodies (Abs) in the presence of soluble rB7-H4 or of acontrol protein (soluble recombinant alpha-folate receptor). T cellsstimulated by anti-CD3 and/or anti-CD28 Abs secreted IFN-γ, expressedthe activation marker CD69 and proliferated as assessed by CFSE labeling(FIGS. 7A-7B). However, the presence of plate-bound rB7-H4 inhibitedIFN-γ secretion (p=0.03 comparing rB7-H4 to control protein), CD69expression and proliferation mediated by CD3 stimulation. While rB7-H4inhibited T-cell activation in response to CD3-stimulation, T cell IFN-γsecretion and proliferation mediated by a combination of anti-CD3 andanti-CD28 mAbs were not significantly inhibited by the presence ofrB7-H4 (p=0.09) (FIGS. 7A-7B). Thus, CD3-mediated T cell stimulation waschosen to functionally characterize the anti-B7-H4 scFvs in the rest ofthe study. FIGS. 7C-7D shows that several anti-B7-H4 scFvs could reverserB7-H4 inhibition of T-cell polyclonal activation (FIGS. 7C and 7D).Anti-B7-H4 scFvs #26 significantly reduced the inhibition of IFN-γ Tcell secretion (p=0.0128 when comparing to untreated condition) but didnot fully overcome B7-H4-mediated inhibition of IFN-γ secretion norreconstituted T cell proliferation (FIG. 7D). In contrast, anti-B7-H4scFvs 3#54 and 3#68 restored T cell IFN-γ secretion (FIG. 7C) andreversed rB7-H4-dependent inhibition of T cell proliferation to normallevels (p=0.0406 for 3#54; p=0.1305 for 3#68) (FIG. 7D). Of note, thepresence of anti-B7-H4 scFv 3#68 in CD3-activated T cells triggeredhigher IFN-γ secretion in the absence of any protein or in the presenceof control protein (FIG. 7C) but did not modify T cell proliferation(FIG. 7D). These results support the hypothesis that scFv interferingwith functional interactions between B7-H4 and B7-H4 putative T cellligand can block B7-H4-dependent T cell inhibition.

Antigen-Specific T Cell Activation is Inhibited by Peptide-Pulsed APCsExpressing B7-H4 and can be Restored by Anti-B7-H4 scFvs

B7-H4 expression has been reported on tumor-infiltrated DCs (Cheng etal., 2011, J Immunoassay Immunochem 32(4):353-64). To model in vitro therole of B7-H4 in a system of antigen presentation that elicits tumorantigen-specific T cell responses, T2 antigen presenting cells (T2 APCs(Salter and Cresswell, 1986, EMBO J 5(5):943-9; Levine et al., 1997, JImmunol, 159(12):5921-30) were transduced with full-length B7-H4 (FIG.8A), and peripheral human T cells were transduced with HER-2 specificTCR (Lanitis E. et al, manuscript in preparation) (FIG. 8B) or MART-1specific TCR (Johnson et al., 2006, J Immunol 177(9):6548-59) (FIG. 8C).As negative control, T2 APC was transduced with GFP. T2 APCs were pulsedwith MART-1 or HER-2 peptides and used to activated TCR transduced Tcells. HER-2 and MART-1 TCR transduced T cells were activated with B7-H4transduced (T2 B7-H4) or GFP transduced (T2), peptide-pulsed T2 APCs,and IFN-γ production was measured. MART-1 peptide was used as anirrelevant peptide for the stimulation of HER-2 TCR T cells and HER-2peptide as an irrelevant peptide for the stimulation of MART-1 TCR Tcells. FIGS. 8B and 8C show that B7-H4 expression on T2 APCsdown-regulated antigen-specific T cell activation in both HER-2 andMART-1 specific T cells (p=0.0362 for HER-2 TCR T cells and p=0.0024 forMART-1), demonstrating that B7-H4-dependent inhibition was notantigen-restricted. B7-H4-mediated inhibition of HER-2 and MART-1specific T cells was partially reversed by anti-B7-H4 scFvs #56 and3#54, but anti-B7-H4 scFv 3#68 could fully overcome B7-H4-mediatedinhibition and restored MART-1 and HER-2 specific T cell responses(p=0.5748 and p=0.2892 respectively for B7-H4⁺ T2 vs. T2) (FIGS. 8D and8E). One Way Anova analysis of the differentially treated GFP⁺ T2conditions resulted in no significant statistical difference (p=0.7893for HER-2 and p=0.2931 for MART-1 TCR T cells) whereas the same analysisfor T2 B7-H4 resulted in statistical significant differences among thedifferent conditions (p=0.0066 for HER-2 and p<0.0001 for MART-1 TCR Tcells). These results confirmed that blocking functional interactionsbetween B7-H4-expressing APCs and T cells using anti-B7-H4 scFv 3#68could overcome B7-H4-dependent T cell inhibition. In addition,functional variations observed between the different anti-B7-H4 scFvssuggested that the mechanism underlying B7-H4-mediated T cell inhibitiondepends on a specific epitope.

Antigen-Specific T Cell Activation is Inhibited by Tumor CellsExpressing B7-H4 and can be Restored by Anti-B7-H4 scFvs

Since B7-H4 can also be expressed at the tumor cell surfaces (FIG. 5),experiments were designed to address whether B7-H4 expressed by tumorcells could inhibit antigen-specific T cell function. Full length B7-H4was transduced in HER-2⁺/HLA-A2⁺ MDA231 breast cancer cell line(positive target) and in HER-2⁺/HLA-A2^(low) 624 melanoma cell line(negative target) (FIG. 9A). These cell lines were assayed forrecognition by HER-2 specific TCR T cells. HER-2 TCR T cells secretedIFN-γ in presence of HER-2 expressing tumor cells (MDA231, dark greybar) but not in presence of HER-2 negative tumor cells (624, light greybar). Furthermore, IFN-γ secretion was substantially inhibited inpresence of B7-H4 transduced MDA231 cells (black bar; p=0.0451) (FIG.9B). As previously observed, anti-B7-H4 scFvs 3#54 and 3#68 couldcompletely restore IFN-γ secretion of HER-2 T cells, thus seeminglybypassing the inhibitory function of B7-H4 transduced MDA231 (p=0.4393for scFv 3#54; p=0.2179 for scFv 3#68) (FIG. 9C). These results furthercorroborated with the hypothesis that blocking B7-H4 can overcomeantigen-specific T cell inhibition mediated by B7-H4 expressed on tumorcell surface.

Antigen-Specific T Cell Activation is Inhibited by B7-H4 ExpressingTumor—Polarized Macrophages in Presence of Peptide-Pulsed T2 APCs andcan be Restored by Anti-B7-H4 scFvs

Macrophages can be polarized towards TAMs by transwell co-culture withtumor cells via exchange of soluble factors (Dangaj et al., 2011, PLoSOne 6(12):e28386; Hagemann et al., 2006, J Immunol 176(8):5023-32).Thus, to study the effect of B7-H4 expressed in trans by tumor-polarizedmacrophages, experiments were designed to set up transwell co-culturesof ovarian cancer cells and in vitro differentiated M1 macrophages. Invitro tumor-polarized macrophages expressed B7-H4 (FIGS. 11A-11C) andare referred herein as B7-H4⁺ TAMs. B7-H4⁺ TAMs were tested for theirability to inhibit MART-specific T cells stimulated with peptide-pulsedT2 APCs. Antigen-specific T cell responses were down-regulated in thepresence of B7-H4⁺ TAMs pulsed with low concentrations of MART peptide(p=0.0287 for 0.0025 μM) (FIG. 10A). Activation and proliferation ofantigen-specific T cells as measured by CFSE staining and CD137expression were more evidently reduced at low peptide concentration(0.0025 μM and 0.05 μM) (As previously observed, anti-B7-H4 scFvs 3#54and 3#68 could completely restore IFN-γ secretion of HER-2 T cells, thusseemingly bypassing the inhibitory function of B7-H4 transduced MDA231(p=0.4393 for scFv 3#54; p=0.2179 for scFv 3#68) (FIG. 9C). Theseresults further corroborated with the hypothesis that blocking B7-H4 canovercome antigen-specific T cell inhibition mediated by B7-H4 expressedon tumor cell surface). Anti-B7-H4 scFvs could reverse T cell inhibitorysignals mediated by B7-H4⁺ TAMs. While anti-B7-H4 scFv #26 restored andsignificantly enhanced T-cell IFN-γ secretion by 1.5 fold (p=0.0144),anti-B7-H4 scFv 3#54 and 3#68 further enhanced T cell IFN-γ productionby >2 folds (p=0.0037 for scFv 3#54 and p=0.0061 for scFv 3#68) (FIG.10B).

B7-H4-Based Targeted Therapy

B7-H4 expression in various types of human cancer tissues andcorrelation with advanced stages, poor patient survival, and tumorinfiltration of T regulatory cells (Zang et al., 2007, Proc Natl AcadSci USA, 104(49):19458-63), makes it a candidate of choice for targetedtherapy. However, B7-H4 expression has been reported to be mainlyintracellular for ovarian cancer cells (Kryczek et al., 2006, J Exp Med203(4):871-81; Choi et al., 2003, J Immunol 171(9):4650-4), thuslimiting targeted therapeutic strategies.

The results presented herein demonstrate that B7-H4 was present at thesurface of Epcam⁺ cancer cells freshly harvested from ascites and solidtumors from ovarian cancer patients, as well as on tumor-infiltratingmonocytes. Consistent with this observation, ovarian cancer xenograftsdeveloped from long term cultured ovarian cancer cell lines upregulatedB7-H4 expression on freshly harvested tumors, and drasticallydownregulated it after short term in vitro culture. Without being boundto any particular theory, these results support the hypothesis thatB7-H4 cell surface expression is regulated by environmental conditions,possibly because B7-H4 expression specifically enhances tumor cellability to escape immune recognition in vivo, but might not benon-essential for cell survival in vitro (Pardoll, 2012, Nat Rev Cancer12(4):252-64). One possible environmental condition able to triggersurface B7-H4 presentation may be hypoxic stress that is of commonoccurrence in the tumor microenvironment. However, hypoxic cultureconditions did not upregulate B7-H4 cell-surface expression in any ofthe ovarian cancer cell lines tested, including OVCAR5. The cytokinemilieu of the tumor microenvironment could be another possible mechanismand in fact, Chen et al. recently reported that macrophage-derived TNF-αinduced B7-H4 cell-surface expression in mouse lung carcinoma (Chen etal., 2012, Cancer Lett 317(1):99-105).

The demonstration that B7-H4 is expressed at the cell surface of tumorand tumor-infiltrating cells opens a new paradigm for simultaneousimmune-modulation of the tumor microenvironment and direct ovariancancer cell eradication using B7-H4-based targeting. To isolaterecombinant antibodies specific for human B7-H4, a novel yeast-displayscFv library derived from B cells of human ovarian cancer ascites wasconstructed. Selected anti-B7-H4 scFvs were then evaluated for theirfunctional ability to reverse B7-H4-mediated T cell inhibition, throughrB7-H4 protein, B7-H4⁺ APCs or B7-H4⁺ tumor cells. The results presentedherein demonstrate that the activation of tumor antigen-TCR specific Tcells was inhibited by the presentation of B7-H4 in cis on APCs or ontumor cells, and in trans on tumor-polarized macrophages. These resultsconfirmed that B7-H4 is a regulatory molecule engaged in negativesignaling that impacts T cell anti-tumor responses. Furthermore, whileB7-H4 transduced APCs impair the activation of antigen-specific T cells,tumor cells transduced to express cell surface B7-H4 can alsosignificantly impair tumor antigen-specific T cell responses. Inaddition, the results presented herein demonstrate that B7-H4-mediatedinhibition in cis or in trans could be partially reversed by anti-B7-H4scFv clone 3#54 and fully restored by anti-B7-H4 scFv 3#68. In fact, intranswell cocultures including blockade with anti-B7-H4 scFv, MART-TCRspecific T cell responses were not only restored in presence of TAMs butfurther enhanced. Without wishing to be bound to any particular theory,it is believed that in addition to blocking B7-H4, anti-B7-H4 alsorestored Th1 proinflammatory environment which could further polarizemacrophages into an M1-like phenotype and stimulate antigen-specific Tcells. Similar results were obtained when using HER-2 TCR specific Tcells.

Two strategies were used to isolate the anti-B7-H4 scFvs, onerepresenting the conventional enrichment of the yeast display library onsoluble recombinant protein by magnetic and flow sortings and a secondone where the yeast display scFv library was further enriched usingcells differentially expressing surface B7-H4. Analysis of the scFvbinding to recombinant B7-H4 by capture ELISA did not highlightdifferences between the differentially isolated scFv categories.However, when testing their functional ability to block B7-H4 mediatedinhibition, the cell-based isolated scFvs showed superior blockadecapacity. This may be due to the epitope each scFv recognizes which incase of scFv 3#68 could be identical with that of the putative B7-H4ligand. It also strongly suggests that isolating scFvs by differentialpanning of the yeast-display scFv library on cells could permit accessto epitopes that are not readily available in recombinant solubleantigens. In addition, the above approach might not be possible in thecase of monoclonal antibody isolation since the host or hybridoma cellsare immunized with soluble antigens.

The antibodies of the invention exhibit activities in blocking B7-H4 invitro and their human sequence could avoid a potential HAMA response andpossible inhibition by endogenous antibodies in vivo. Notably theserecombinant antibodies have a smaller size than conventional antibodiesfavoring their potential binding to target cells expressing B7-H4 evenin areas small penetration.

Targeting B7-H4 can simultaneously modify critical components of thetumor microenvironment, including tumor-associated macrophages, DCs andtumor cells. Blocking the inhibitory signals mediated by B7-H4 canpotentiate T cell anti-tumor responses and could also decrease tumorendothelial T cell barrier because B7-H4 expression on tumor endothelialcells has also been observed in RCC tissues (Krambeck et al., 2006, ProcNatl Acad Sci USA 103(27):10391-6). Targeting immune checkpointmolecules such as CTLA-4 and PD-1 has elicited clinical resultsespecially in patients with pre-existing immune responses. Ovariancancer is a disease largely involving immune circuits that could predictbetter patient survival (Zhang et al., 2003, N Engl J Med 348(3):203-13)or poorer outcome (Kryczek et al., 2007, Cancer Res 67(18):8900-5;Curiel et al., 2004, Nat Med 10(9):942-9).

Example 3 B7-H4-Specific CAR

The following experiments were performed to validate a chimeric antigenreceptor to redirect T-cells against B7-H4-expressing targets using theantibodies of the present invention.

B7-H4-Specific CAR Construction

Anti-B7H4 scFv clones #56, #26, 3#54, and 3#68 were selected toconstruct B7-H4-specific CAR for the reason of relative high antigenbinding affinity among the identified scFvs. The lentiviralCAR-expressed vector presently used in the experiment has been optimizedbefore (Carpenito, et al., 2009, Proc Natl Acad Sci USA 106:3360-3365)and constitute a CD8a hinge and transmembrane region, followed by a CD3ξsignaling moiety and in tandem with the CD28 intracellular signalingmotif. The cDNA of the respective scFv was sub-cloned into theselentiviral-CAR vectors. Further, these vectors were transformed into293T cells and western blot probed to CD3ξ confirmed successfulexpression by these vectors.

For effective lentiviral transduction, human T lymphocytes fromperipheral blood were activated by CD3/CD28 beads. To test thetransduction efficiency, T cells were transduced with GFP-expressedlentiviral vector, and the stable consistent GFP expression can beobserved after 5 days transduction. FIG. 13 shows that creation of 4anti-B7-H4 CARs of different binding affinities (Low to high 26, 56, 68,54) in first or second generation constructs (+/−costim). CARs withdifferent B7-H4 single chains exhibited varying affinity to human B7-H4antigen. The B7-H4 CARs bind both mouse and human B7-H4 proteins (FIG.14).

3E11-CAR+ T Cells Showed B7-H4-Specific Cytotoxicity In Vitro

Engineered T cells were cocultured with B7-H4+ ovarian cancer cells todetermine the effects of antigen specific cytotoxicity. T cells weretransduced by lentiviral vector of the respective B7-H4 scFv-28z (CD28and CD3zeta) and applied to cytotoxicity assays as measured by theconcentration of IFN-γ (FIGS. 15A-15B). As shown in FIG. 15A, T cellstransduced with B7-H4-28z have significant cellular lysis of OVCAR3,while no lysis effects on CD19-28z transduced T cells. CARs 68, 54 and26 reacted against a human cancer cell line expressing B7-H4 on thesurface (A1847), but not against a B7-H4 negative line (c30) (FIG. 15A).FIG. 15B demonstrates that CAR 68 reacts against a variety of humancancer cells expressing B7-H4 on the surface. CAR 56 has low activityagainst an EBV cancer cell line.

These results demonstrate that the CAR transduced T cells can be used totarget B7-H4 expressing tumors as a type of T cell-based immunotherapyof ovarian cancer. The results presented herein provide a specific andhuman-sourced scFv for CAR-transduced T cells-based immunotherapy.

Experiments were conducted to assess whether B7H4 CAR transduced T cellsare reactive against macrophages that express B7H4. FIG. 16Ademonstrates that B7H4 CAR (e.g., 68-28z) transduced T cells arereactive against macrophages that express different levels of B7H4 asmeasured by the concentration of IFN-γ. CAR 68 reacted against humanmacrophages expressing high or low levels of surface B7-H4, andtherefore are suited for targeting immunosuppressive tumor associatedmacrophages (TAMs).

The results presented herein demonstrate that B7H4 CARs are able torespond to tumor cell lines expressing endogenous B7H4 antigen,including both solid and lymphoma cell lines. Most robust level ofrecognition was observed by 68 B7H4 CAR, comparable to wellcharacterized CD19 CAR against B cell lymphoma cell line (FIG. 16B).B7H4 CARs also recognized macrophages expressing both low and highlevels of B7H4, which can aid in reducing B7-H4+ tumor associatedmacrophages in the tumor microenvironment.

The next set of experiments was conducted to assess whether B7-H4 CARscan be inhibited by the addition of a corresponding B7-H4 scFv (FIG.17A). All T cells were stimulated with anti-CD3 OKT-3 Ab (1 ug/mL) inthe presence of increasing concentrations of B7-H4 protein. Values werenormalized to anti-CD3 Ab plus control protein (folate receptor) atequivalent concentrations (**Normalized to IFN-γ % with OKT3+αFolate(irrelavent) Ag as 100% activity). Specific inhibition of 68 B7H4 CARIFN-γ secretion was observed by the addition of 68scFv (FIG. 17B). Itwas also observed that 68 B7H4 CAR IFN-γ secretion in response to B7H4Ag is not significantly reduced by B7H4 inhibitory signaling. CAR 68 Tcells were found to be resistant to inhibition delivered by negativeB7-H4 signals, whereas conventional (e.g., CD19 specific) CARs for otherantigens are not.

Also, the addition of 68scFv in the presence of CD3 led to specificinhibition of 68 B7H4 CAR IFN-γ secretion and specific moderate rescueof 56 B7H4 CAR IFN-γ secretion (FIG. 18). For example, CAR 68 T cellswere found to be specific for B7-H4 since their activity was blocked bypre-blocking B7-H4 with soluble anti-B7-H4 scFv 68 before culture).

It was also observed that 68scFv B7H4 CAR MIP1a cytokine secretion isnot diminished as a result of B7H4 CAR-B7H4 Ag signaling (FIG. 19).

The results presented here demonstrate that 68 B7H4 CAR IFNγ and MIP1asecretion in response to B7H4 Ag is not significantly reduced by B7H4inhibitory signaling; however, CARs of other specificity are. Theaddition of soluble #68scFv led to specific masking of B7-H4 and thusinhibited 68 B7H4 CAR IFNγ and MIP1a secretion.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1.-2. (canceled)
 3. An isolated polypeptide encoding a human anti-B7-H4antibody or a fragment thereof comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-4.
 4. Thepolypeptide of claim 3, wherein the antibody or fragment thereofcomprises a fragment selected from the group consisting of an Fabfragment, an F(ab′)₂ fragment, an Fv fragment, and a single chain Fv(scFv). 5.-30. (canceled)
 31. The polypeptide of claim 3, wherein theantibody or fragment thereof binds B7-H4.
 32. The polypeptide of claim31, wherein the antibody or fragment thereof binds B7-H4 on ovariantumor cells.
 33. The polypeptide of claim 3 further comprising theantibody or fragment thereof operably linked to a biologically activeagent.
 34. The polypeptide of claim 33, wherein the biologically activeagent is selected from the group consisting of a cytotoxic agent, achemotherapeutic agent, a cytokine, a growth inhibitory agent, ananti-hormonal agent, a kinase inhibitor, an anti-angiogenic agent, acardioprotectant, a toxin, a radioisotope, and a combination thereof.35. The polypeptide of claim 3 further comprising the antibody orfragment thereof operably linked to a labeling agent.
 36. Thepolypeptide of claim 36, wherein the labeling agent is selected from thegroup consisting of a nanoparticle quantum dot, a fluorophore, acyanide, a radioactively-labeled peptide, a magnetic nanoparticle, achromophore, and a localization marker.